Crystal modifications of odevixibat

ABSTRACT

The present invention relates to crystal modifications of 1,1-dioxo-3,3-dibutyl-5-phenyl-7-methylthio-8-(N—{(R)-α-[N—((S)-1-carboxypropyl)carbamoyl]-4-hydroxybenzyl}carbamoylmethoxy)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine (odevixibat), more specifically crystal modifications 1 and 2 of odevixibat. The invention also relates to a process for the preparation of crystal modification 1 of odevixibat, to a pharmaceutical composition comprising crystal modification 1, and to the use of this crystal modification in the treatment of various conditions as described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation under 35 U.S.C. § 111(a) ofInternational Application No. PCT/SE2019/050602, filed Jun. 20, 2019,which claims priority to Swedish Application No. 1850761-6, filed Jun.20, 2018, and to Swedish Application No. 1850762-4, filed Jun. 20, 2018,the disclosures of which are incorporated by reference herein in theirentireties.

TECHNICAL FIELD

The present invention relates to crystal modifications of1,1-dioxo-3,3-dibutyl-5-phenyl-7-methylthio-8-(N—{(R)-α-[N—((S)-1-carboxypropyl)carbamoyl]-4-hydroxybenzyl}carbamoylmethoxy)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine(odevixibat), more specifically crystal modifications 1 and 2 ofodevixibat. The invention also relates to a process for the preparationof crystal modification 1 of odevixibat, to a pharmaceutical compositioncomprising crystal modification 1, and to the use of this crystalmodification in the treatment of various conditions as described herein.

BACKGROUND

The compound1,1-dioxo-3,3-dibutyl-5-phenyl-7-methylthio-8-(N—{(R)-α-[N—((S)-1-carboxypropyl)carbamoyl]-4-hydroxybenzyl}carbamoylmethoxy)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine(odevixibat; also known as A4250) is disclosed in WO 03/022286. Thestructure of odevixibat is shown below.

As an inhibitor of the ileal bile acid transporter (IBAT) mechanism,odevixibat inhibits the natural reabsorption of bile acids from theileum into the hepatic portal circulation. Bile acids that are notreabsorbed from the ileum are instead excreted into the faeces. Theoverall removal of bile acids from the enterohepatic circulation leadsto a decrease in the level of bile acids in serum and the liver.Odevixibat, or a pharmaceutically acceptable salt thereof, is thereforeuseful in the treatment or prevention of diseases such as dyslipidemia,constipation, diabetes and liver diseases, and especially liver diseasesthat are associated with elevated bile acid levels.

According to the experimental section of WO 03/022286, the last step inthe preparation of odevixibat involves the hydrolysis of a tert-butylester under acidic conditions. The crude compound was obtained byevaporation of the solvent under reduced pressure followed bypurification of the residue by preparative HPLC (Example 29). Nocrystalline material was identified.

Amorphous materials may contain high levels of residual solvents, whichis highly undesirable for materials that should be used aspharmaceuticals. Also, because of their lower chemical and physicalstability, as compared with crystalline material, amorphous materialsmay display faster decomposition and may spontaneously form crystalswith a variable degree of crystallinity. This may result inunreproducible solubility rates and difficulties in storing and handlingthe material. In pharmaceutical preparations, the active pharmaceuticalingredient (API) is for that reason preferably used in a highlycrystalline state. Thus, there is a need for crystal modifications ofodevixibat having improved properties with respect to stability, bulkhandling and solubility. In particular, it is an object of the presentinvention to provide a stable crystal modification of odevixibat thatdoes not contain high levels of residual solvents, that has improvedchemical stability and can be obtained in high levels of crystallinity.

SUMMARY OF THE INVENTION

The invention provides crystal modifications of odevixibat. In a firstaspect, the crystal modification is a crystalline hydrate of odevixibat.This crystalline hydrate is a channel hydrate, which may contain up to 2moles of water associated with the crystal per mole of odevixibat. Theamount of water calculated herein excludes water adsorbed to the surfaceof the crystal. In one embodiment, the crystalline hydrate is asesquihydrate, i.e., contains about 1.5 moles of water associated withthe crystal per mole of odevixibat. In another aspect, which may berelated to the first aspect, the invention provides crystal modification1 of odevixibat. Crystal modification 1 is a stable crystalline hydratewhich at 30% relative humidity (RH) contains about 1.5 moles of waterper mole of odevixibat.

In another aspect, the invention provides a dihydrate-disolvate ofodevixibat. This mixed solvate can exist as different isostructuralsolvates and may comprise methanol, ethanol, 2-propanol, acetone,acetonitrile, 1,4-dioxane, DMF or DMSO as the organic solvent. When themixed solvate is dried, it loses its solvate molecules and transformsinto crystal modification 1 of odevixibat. In another aspect, which maybe related to this aspect, the invention provides crystal modifications2A, 2B and 2C of odevixibat, herein collectively referred to as crystalmodification 2 of odevixibat. Upon drying, crystal modification 2 losesits organic solvent molecules and generates crystal modification 1 ofodevixibat.

The invention further provides the use of crystal modification 1 ofodevixibat in the treatment of a condition described herein, apharmaceutical composition comprising crystal modification 1 ofodevixibat, as well as a process for the preparation of crystalmodification 1 of odevixibat.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the X-ray powder diffractogram of dried crystalmodification 1.

FIG. 2 shows the X-ray powder diffractogram of an overhydrated sample ofcrystal modification 1.

FIG. 3 shows the drying of crystal modification 1, with the X-ray powderdiffractogram of an overhydrated sample of crystal modification 1 at thebottom and of a dried sample at the top (2θ range 5-13°).

FIG. 4 shows the drying of crystal modification 1, with the X-ray powderdiffractogram of an overhydrated sample of crystal modification 1 at thebottom and of a dry sample at the top (2θ range 18-25°).

FIG. 5 shows the transformation from crystal modification 2 (bottom), asobtained from a mixture of ethanol (60-80% v/v) and water (20-40% v/v),to crystal modification 1 (top) via crystal modification 12 (middle).

FIG. 6 shows the X-ray powder diffractogram of crystal modification 2A,as obtained from a mixture of ethanol and water (70:30% v/v).

FIG. 7 shows the X-ray powder diffractogram of crystal modification 2A,as obtained from a mixture of acetone and water (50:50% v/v).

FIG. 8 shows the X-ray powder diffractogram of crystal modification 2A,as obtained from a mixture of 2-propanol and water (50:50% v/v).

FIG. 9 shows the X-ray powder diffractogram of crystal modification 2A,as obtained from a mixture of 1,4-dioxane and water (50:50% v/v).

FIG. 10 shows the X-ray powder diffractogram of crystal modification 2B,as obtained from methanol. The water that is necessary for form 2 tocrystallize was obtained from the air, as a result of the hygroscopicityof methanol.

FIG. 11 shows the X-ray powder diffractogram of crystal modification 2B,as obtained from a mixture of acetonitrile and water (40:60% v/v).

FIG. 12 shows the X-ray powder diffractogram of crystal modification 2C,as obtained from a mixture of DMSO and water (50:50% v/v).

FIG. 13 shows the thermogravimetric analysis (TGA) mass change plot forcrystal modification 1.

FIG. 14 shows the thermogravimetric analysis (TGA) mass change plot forcrystal modification 2 produced by exposure of crystal modification 1 tothe vapor phase of a mixture of ethanol and water FIG. 15 shows thedynamic vapour sorption (DVS) mass change plot for crystal modification1.

FIG. 16 shows the DSC trace of a sample of odevixibat with about 50%crystalline fraction (after pre-heating and cooling).

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein relates to crystal modifications thatwere discovered in extensive studies on odevixibat. It has been observedthat odevixibat can crystallize from a variety of organic solvents (ormixtures of solvents) by incorporating solvate molecules in itsstructure, thereby forming various solvates or mixed solvates. Whilemost of these (mixed) solvates are unstable in air and become amorphousupon drying, it has surprisingly been discovered that certain mixedsolvates of odevixibat could be dried and transformed into a stablecrystalline form of odevixibat. It is remarkable that this stable form,hereinafter referred to as crystal modification 1 of odevixibat, can beformed from different mixed solvates of odevixibat.

Thus, in a first aspect, the invention relates to crystal modification 1of odevixibat. This stable crystal modification can be obtained from aslurry of odevixibat in a mixture of water and an organic solvent suchas ethanol. Under these conditions, a mixed solvate containing about twomoles of water and about one to about three, such as about two to aboutthree, moles of ethanol per mole of odevixibat (e.g., adihydrate-diethanolate or a dihydrate-triethanolate) is initiallyformed. In some embodiments, this mixed solvate is referred to ascrystal modification 2. When the mixed solvate is dried, it loses itsorganic solvent molecules and becomes crystal modification 1. While notwishing to be bound by theory, it is believed that the solvent moleculescan be removed without dissolution and recrystallization of thecrystals.

Crystal modification 1 contains void volumes that are capable ofcontaining up to about 2 moles of water associated with the crystal permole of odevixibat, depending on the relative humidity. This form istherefore formally a channel hydrate. At about 30% relative humidity,however, crystal modification 1 contains a substantially stoichiometricamount of about 1.5 moles of water per mole of organic compound and isthus a sesquihydrate. The substantially stoichiometric amount of wateris considered advantageous, as the water content of the crystals remainssubstantially constant even with humidity changes within the normalrelative humidity range of about 30% to about 70% RH.

Indeed, at normal humidities, such as between about 30 and about 70% RH,crystal modification 1 exhibits relatively low hygroscopicity.

In one embodiment, the invention relates to crystal modification 1 ofodevixibat having an X-ray powder diffraction (XRPD) pattern, obtainedwith CuKα1-radiation, with at least specific peaks at °2θ positions5.6±0.2, 6.7±0.2 and/or 12.1±0.2.

In a specific embodiment thereof, the invention relates to crystalmodification 1 having an XRPD pattern, obtained with CuKα1-radiation,with specific peaks at °2θ positions 5.6±0.2, 6.7±0.2 and 12.1±0.2 andone or more of the characteristic peaks: 4.1±0.2, 4.6±0.2, 9.3±0.2,9.4±0.2 and 10.7±0.2.

In a more specific embodiment thereof, the invention relates to crystalmodification 1 having an XRPD pattern, obtained with CuKα1-radiation,with specific peaks at °2θ positions 4.6±0.2, 5.6±0.2, 6.7±0.2, 9.3±0.2,9.4±0.2 and 12.1±0.2.

In a yet more specific embodiment thereof, the invention relates tocrystal modification 1 having an XRPD pattern, obtained withCuKα1-radiation, with characteristic peaks at °2θ positions 4.1±0.2,4.6±0.2, 5.6±0.2, 6.7±0.2, 9.3±0.2, 9.4±0.2, 10.7±0.2 and 12.1±0.2, andone or more of 8.1±0.2, 8.6±0.2, 13.4±0.2, 13.8±0.2, 13.9±0.2, 16.6±0.2,17.3±0.2, 17.7±0.2, 18.3±0.2, 18.9±0.2, 19.4±0.2, 19.7±0.2, 20.5±0.2,20.8±0.2, 21.6±0.2, 23.2±0.2, 24.3±0.2, 29.8±0.2 and 30.6±0.2.

In a yet even more specific embodiment thereof, the invention relates tocrystal modification 1 having an XRPD pattern, obtained withCuKα1-radiation, with characteristic peaks at °2θ positions 4.1±0.2,4.6±0.2, 5.6±0.2, 6.7±0.2, 8.1±0.2, 8.6±0.2, 9.3±0.2, 9.4±0.2, 10.7±0.2,12.1±0.2, 13.4±0.2, 13.8±0.2, 13.9±0.2, 16.6±0.2, 17.3±0.2, 17.7±0.2,18.3±0.2, 18.9±0.2, 19.4±0.2, 19.7±0.2, 20.5±0.2, 20.8±0.2, 21.6±0.2,23.2±0.2, 24.3±0.2, 29.8±0.2 and 30.6±0.2.

In a particular embodiment, the invention relates to crystalmodification 1 having an XRPD pattern, obtained with CuKα1-radiation,substantially as shown in FIG. 1.

Whereas crystal modification 1 is a sesquihydrate containing about 3.5%(w/w) water at about 30% relative humidity (based on the total crystalweight), it has been observed that the crystal can take up an additional1.5% (w/w) water when the humidity is increased up to 95% RH. Thesorption and desorption of this additional water is fully reversible(see e.g. Example 10). The additional water may be adsorbed on thesurface or may further fill the channels of the structure. In someembodiments, the term “overhydrated” refers to crystal modification 1containing from about 1.5 to about 4 moles of water per mole ofodevixibat, such as from about 1.5 to about 3.5, or such as from about1.5 to 3, or such as from about 1.5 to about 2.5, or such as from about1.5 to about 2 moles of water per mole of odevixibat. In someembodiments, the term “overhydrated” refers to crystal modification 1containing from about 2 to about 4 moles of water per mole ofodevixibat, such as from about 2 to about 3.5, or such as from about 2to about 3, or such as from about 2 to 2.5 moles of water per mole ofodevixibat.

It has been observed that the XRPD pattern of overhydrated crystalmodification 1 slightly changes when it is dried, e.g. at 50° C. invacuum. A small shift of peaks is most clearly seen in the 20 ranges5-13° and 18-25°, as shown in FIGS. 3 and 4, respectively. Exposing thedried modification to elevated relative humidity, such as up to 95% RH,makes the XRPD pattern of the overhydrated modification appear again.The peak shifts are a result of the unit cell volume changes, whichoccur as water molecules go in and out of the crystal structure.

Therefore, in another embodiment, the invention relates to overhydratedcrystal modification 1 having an X-ray powder diffraction (XRPD)pattern, obtained with CuKα1-radiation, with at least specific peaks at°2θ positions 5.7±0.2, 6.7±0.2 and/or 12.0±0.2.

In certain embodiments, the invention relates to overhydrated crystalmodification 1 having an XRPD pattern, obtained with CuKα1-radiation,with specific peaks at °2θ positions 5.7±0.2, 6.7±0.2 and 12.0±0.2 andone or more of the characteristic peaks: 4.0±0.2, 9.4±0.2, 9.6±0.2 and10.8±0.2.

In a more particular embodiment, the invention relates to overhydratedcrystal modification 1 having an XRPD pattern, obtained withCuKα1-radiation, with specific peaks at °2θ positions 4.0±0.2, 5.7±0.2,6.7±0.2, 9.4±0.2, 9.6±0.2, 10.8±0.2 and 12.1±0.2.

In a further embodiment, the invention relates to overhydrated crystalmodification 1 having an XRPD pattern, obtained with CuKα1-radiation,with characteristic peaks at °2θ positions 4.0±0.2, 5.7±0.2, 6.7±0.2,9.4±0.2, 9.6±0.2, 10.8±0.2 and 12.1±0.2, and one or more of 4.7±0.2,8.0±0.2, 8.6±0.2, 13.3±0.2, 14.1±0.2, 15.3±0.2, 16.5±0.2, 17.3±0.2,19.3±0.2, 19.7±0.2, 19.9±0.2, 20.1±0.2, 20.8±0.2, 21.7±0.2, 23.6±0.2,26.2±0.2, 26.5±0.2, 28.3±0.2 and 30.9±0.2.

In a yet further embodiment, the invention relates to overhydratedcrystal modification 1 having an XRPD pattern, obtained withCuKα1-radiation, with characteristic peaks at °2θ positions 4.0±0.2,4.7±0.2, 5.7±0.2, 6.7±0.2, 8.0±0.2, 8.6±0.2, 9.4±0.2, 9.6±0.2, 10.8±0.2,12.1±0.2, 13.3±0.2, 14.1±0.2, 15.3±0.2, 16.5±0.2, 17.3±0.2, 19.3±0.2,19.7±0.2, 19.9±0.2, 20.1±0.2, 20.8±0.2, 21.7±0.2, 23.6±0.2, 26.2±0.2,26.5±0.2, 28.3±0.2 and 30.9±0.2.

In yet another embodiment, the invention relates to overhydrated crystalmodification 1 of odevixibat having an XRPD pattern, obtained withCuKα1-radiation, substantially as shown in FIG. 2.

In some embodiments, the crystallinity of crystal modification 1 isgreater than about 99%. The crystallinity may be measured byDifferential Scanning Calorimetry (DSC) methods, e.g. as disclosed inthe experimental section.

Crystal modification 1 has several advantages over amorphous odevixibat.The relatively low hygroscopicity of crystal modification 1 at normalhumidities, such as 30-70% RH, facilitates the handling and storing ofodevixibat. Additionally, crystal modification 1 does not contain highlevels of residual solvents. In contrast, it has been observed thatbatches of crude, amorphous odevixibat can contain residual solvents(such as formic acid) at levels that exceed the regulatory limits byfar. Stability experiments have further shown that crystal modification1 of odevixibat displays a higher chemical stability than amorphousodevixibat.

Crystal modification 1 may possess one or more additional advantages,such as a higher physical and thermodynamic stability than amorphousodevixibat; a more reproducible solubility than amorphous odevixibat; oran improved ability to process into a formulation. Such properties arehighly relevant for pharmaceutical formulations of odevixibat.

In a second aspect, the invention relates to crystal modification 2 ofodevixibat. It has been discovered that crystal modification 2 may beobtained not only from a mixture of ethanol and water, as describedabove, but also from methanol and certain other mixtures of solvent andwater, including mixtures of methanol and water, 2-propanol and water,acetone and water, acetonitrile and water, 1,4-dioxane and water, DMFand water and DMSO and water. Crystal modification 2 is a mixed solvate,containing about two moles of water and about one to about three molesof organic solvent per mole of odevixibat. In some embodiments, themixed solvate includes about 1.7 to about 2.3, about 1.8 to about 2.2,about 1.9 to about 2.1 or about 1.95 to about 2.05 moles of waterassociated with each mole of odevixibat in a crystal (excluding anywater that may be adsorbed to the surface of the crystal).

Interestingly, the XRPD patterns for the crystal modifications obtainedfrom these different mixtures are essentially the same (see FIGS. 6-12).It is therefore believed that crystal modification 2 can exist asdifferent isostructural solvates (also known as isomorphic solvates). Inthese isostructural solvates, crystal modification 2 accommodatesdifferent solvents (as a mixture with water). The presence of differentsolvents causes small volume changes to the unit cell but does nototherwise result in any significant distortion of the crystal structureof crystal modification 2. Nevertheless, the XRPD patterns for theisostructural solvates may be slightly different. Three similar, yetslightly different forms of crystal modification 2 are herein referredto as crystal modifications 2A, 2B and 2C, and collectively as “crystalmodification 2”. Significantly, it has been found that upon drying,crystal modifications 2A, 2B and 2C can form crystal modification 1,regardless of the solvent mixture from which crystal modification 2 wascrystallized.

In a first embodiment, the crystalline mixed solvate is crystalmodification 2A, as obtained from a mixture of ethanol and water,acetone and water, 1,4-dioxane and water, DMF and water or 2-propanoland water, having an X-ray powder diffraction (XRPD) pattern, obtainedwith CuKα1-radiation, with at least specific peaks at °2θ positions5.0±0.2, 5.1±0.2 and/or 11.8±0.2.

In a specific embodiment thereof, the invention relates to crystalmodification 2A, as obtained from a mixture of ethanol and water,acetone and water, 1,4-dioxane and water, DMF and water or 2-propanoland water, having an XRPD pattern, obtained with CuKα1-radiation, withspecific peaks at °2θ positions 5.0±0.2, 5.1±0.2 and 11.8±0.2 and one ormore of the characteristic peaks: 6.4±0.2, 6.6±0.2 and 9.5±0.2.

In a more specific embodiment thereof, the invention relates to crystalmodification 2A, as obtained from a mixture of ethanol and water,acetone and water, 1,4-dioxane and water, DMF and water or 2-propanoland water, having an XRPD pattern, obtained with CuKα1-radiation, withspecific peaks at °2θ positions 5.0±0.2, 5.1±0.2, 6.4±0.2, 6.6±0.2,9.5±0.2 and 11.8±0.2.

In a yet more specific embodiment thereof, the invention relates tocrystal modification 2A, as obtained from a mixture of ethanol andwater, having an XRPD pattern, obtained with CuKα1-radiation, withcharacteristic peaks at °2θ 5.0±0.2, 5.1±0.2, 6.4±0.2, 6.6±0.2, 9.5±0.2and 11.8±0.2, and one or more of 5.9±0.2, 8.8±0.2, 9.8±0.2, 10.1±0.2,11.0±0.2, 11.2±0.2, 11.4±0.2, 12.7±0.2, 13.9±0.2, 14.7±0.2, 15.1±0.2,15.8±0.2, 16.3±0.2, 17.2±0.2, 17.9±0.2, 19.7±0.2, 20.2±0.2, 20.7±0.2,21.3±0.2, 22.1±0.2, 22.5±0.2, 22.9±0.2, 23.2±0.2, 23.6±0.2, 24.0±0.2,24.1±0.2, 24.7±0.2, 25.3±0.2, 26.7±0.2, 26.9±0.2, 29.8±0.2, 30.4±0.2,30.8±0.2 and 31.6±0.2.

In a yet even more specific embodiment thereof, the invention relates tocrystal modification 2A, as obtained from a mixture of ethanol andwater, having an XRPD pattern, obtained with CuKα1-radiation, withcharacteristic peaks at °2θ positions 5.0±0.2, 5.1±0.2, 5.9±0.2,6.4±0.2, 6.6±0.2, 8.8±0.2, 9.5±0.2, 9.8±0.2, 10.1±0.2, 11.0±0.2,11.2±0.2, 11.4±0.2, 11.8±0.2, 12.7±0.2, 13.9±0.2, 14.7±0.2, 15.1±0.2,15.8±0.2, 16.3±0.2, 17.2±0.2, 17.9±0.2, 19.7±0.2, 20.2±0.2, 20.7±0.2,21.3±0.2, 22.1±0.2, 22.5±0.2, 22.9±0.2, 23.2±0.2, 23.6±0.2, 24.0±0.2,24.1±0.2, 24.7±0.2, 25.3±0.2, 26.7±0.2, 26.9±0.2, 29.8±0.2, 30.4±0.2,30.8±0.2 and 31.6±0.2.

In one particular embodiment, the invention relates to crystalmodification 2A, as obtained from a mixture of ethanol and water, havingan XRPD pattern, obtained with CuKα1-radiation, substantially as shownin FIG. 6.

In another particular embodiment, the invention relates to crystalmodification 2A, as obtained from a mixture of acetone and water, havingan XRPD pattern, obtained with CuKα1-radiation, substantially as shownin FIG. 7.

In yet another particular embodiment, the invention relates to crystalmodification 2A, as obtained from a mixture of 2-propanol and water,having an XRPD pattern, obtained with CuKα1-radiation, substantially asshown in FIG. 8.

In yet another particular embodiment, the invention relates to crystalmodification 2A, as obtained from a mixture of 1,4-dioxane and water,having an XRPD pattern, obtained with CuKα1-radiation, substantially asshown in FIG. 9.

In a second embodiment, the crystalline mixed solvate is crystalmodification 2B, as obtained from methanol or from a mixture of methanoland water or acetonitrile and water, having an X-ray powder diffraction(XRPD) pattern, obtained with CuKα1-radiation, with at least specificpeaks at °2θ positions 4.8±0.2, 5.1±0.2 and/or 11.6±0.2.

In a specific embodiment, the invention relates to crystal modification2B, as obtained from methanol or from a mixture of methanol and water oracetonitrile and water, having an XRPD pattern, obtained withCuKα1-radiation, with specific peaks at °2θ positions 4.8±0.2, 5.1±0.2and 11.6±0.2 and one or more of the characteristic peaks: 6.2±0.2,6.7±0.2, 9.5±0.2 and 20.3±0.2.

In a more specific embodiment thereof, the invention relates to crystalmodification 2B, as obtained from methanol or from a mixture of methanoland water or acetonitrile and water, having an XRPD pattern, obtainedwith CuKα1-radiation, with specific peaks at °2θ positions 4.8±0.2,5.1±0.2, 6.2±0.2, 6.7±0.2, 9.5±0.2, 11.6±0.2 and 20.3±0.2.

In a yet more specific embodiment thereof, the invention relates tocrystal modification 2B, obtained from methanol and water, having anXRPD pattern, obtained with CuKα1-radiation, with characteristic peaksat °2θ positions 4.8±0.2, 5.1±0.2, 6.2±0.2, 6.7±0.2, 9.5±0.2, 11.6±0.2and 20.3±0.2, and one or more of 5.8±0.2, 8.7±0.2, 9.7±0.2, 10.1±0.2,10.7±0.2, 11.5±0.2, 13.4±0.2, 13.5±0.2, 14.4±0.2, 14.5±0.2, 15.2±0.2,16.5±0.2, 16.8±0.2, 19.4±0.2, 20.6±0.2, 21.2±0.2, 21.5±0.2, 23.8±0.2,23.9±0.2, 25.4±0.2, 26.3±0.2, 26.7±0.2, 30.1±0.2 and 30.6±0.2.

In a yet even more specific embodiment thereof, the invention relates tocrystal modification 2B, obtained from methanol and water, having anXRPD pattern, obtained with CuKα1-radiation, with characteristic peaksat °2θ positions 4.8±0.2, 5.1±0.2, 5.8±0.2, 6.2±0.2, 6.7±0.2, 8.7±0.2,9.5±0.2, 9.7±0.2, 10.1±0.2, 10.7±0.2, 11.5±0.2, 11.6±0.2, 13.4±0.2,13.5±0.2, 14.4±0.2, 14.5±0.2, 15.2±0.2, 16.5±0.2, 16.8±0.2, 19.4±0.2,20.3±0.2, 20.6±0.2, 21.2±0.2, 21.5±0.2, 23.8±0.2, 23.9±0.2, 25.4±0.2,26.3±0.2, 26.7±0.2, 30.1±0.2 and 30.6±0.2.

In one particular embodiment, the invention relates to crystalmodification 2B, as obtained from methanol, having an XRPD pattern,obtained with CuKα1-radiation, substantially as shown in FIG. 10.

In another particular embodiment, the invention relates to crystalmodification 2B, as obtained from a mixture of acetonitrile and water,having an XRPD pattern, obtained with CuKα1-radiation, substantially asshown in FIG. 11.

In a third embodiment, the invention relates to crystal modification 2C,as obtained from a mixture of DMSO and water, having an X-ray powderdiffraction (XRPD) pattern, obtained with CuKα1-radiation, with at leastspecific peaks at °2θ positions 5.0±0.2, 6.2±0.2, 9.4±0.2 and/or23.9±0.2.

In a specific embodiment thereof, the invention relates to crystalmodification 2C, as obtained from a mixture of DMSO and water, having anXRPD pattern, obtained with CuKα1-radiation, with specific peaks at °2θpositions 5.0±0.2, 6.2±0.2, 9.4±0.2 and 23.9±0.2 and one or more of thecharacteristic peaks: 11.5±0.2, 19.5±0.2 and 20.2±0.2.

In a more specific embodiment thereof, the invention relates to crystalmodification 2C, as obtained from a mixture of DMSO and water, having anXRPD pattern, obtained with CuKα1-radiation, with specific peaks at °2θpositions 5.0±0.2, 6.2±0.2, 9.4±0.2, 11.5±0.2, 19.5±0.2, 20.2±0.2 and23.9±0.2.

In a yet more specific embodiment thereof, the invention relates tocrystal modification 2C, as obtained from a mixture of DMSO and water,having an XRPD pattern, obtained with CuKα1-radiation, withcharacteristic peaks at °2θ positions 5.0±0.2, 6.2±0.2, 9.4±0.2,11.5±0.2, 19.5±0.2, 20.2±0.2 and 23.9±0.2, and one or more of 4.9±0.2,5.8±0.2, 6.6±0.2, 8.6±0.2, 9.7±0.2, 10.0±0.2, 10.8±0.2, 13.5±0.2,15.1±0.2, 17.7±0.2, 17.9±0.2, 19.0±0.2, 19.3±0.2, 20.7±0.2, 21.1±0.2,21.2±0.2, 21.2±0.2, 22.8±0.2, 25.3±0.2, 26.6±0.2, 27.3±0.2, 27.4±0.2,28.6±0.2, 30.1±0.2 and 30.2±0.2.

In a yet even more specific embodiment thereof, the invention relates tocrystal modification 2C, as obtained from a mixture of DMSO and water,having an XRPD pattern, obtained with CuKα1-radiation, withcharacteristic peaks at °2θ positions 4.9±0.2, 5.0±0.2, 5.8±0.2,6.2±0.2, 6.6±0.2, 8.6±0.2, 9.4±0.2, 9.7±0.2, 10.0_0.2, 10.8±0.2,11.5±0.2, 13.5±0.2, 15.1±0.2, 17.7±0.2, 17.9±0.2, 19.0±0.2, 19.3±0.2,19.5±0.2, 20.2±0.2, 20.7±0.2, 21.1±0.2, 21.2±0.2, 21.3±0.2, 22.8±0.2,23.9±0.2, 25.3±0.2, 26.6±0.2, 27.3±0.2, 27.4±0.2, 28.6±0.2, 30.1±0.2 and30.2±0.2.

In one particular embodiment, the invention relates to crystalmodification 2C, as obtained from a mixture of DMSO and water, having anXRPD pattern, obtained with CuKα1-radiation, substantially as shown inFIG. 12.

As will be understood from the above, the isolation and characterizationof stable crystal modification 1 was not straightforward. Even though itis a hydrate, crystal modification 1 cannot be obtained directly bycrystallization from water. In some embodiments, crystal modification 1is obtained indirectly, e.g. by isolating and drying crystalmodification 2, which is formed by crystallization of odevixibat frommixtures of water and certain organic solvents. In some embodiments,crystal modification 1 is obtained from crystal modification 2 afterevaporation of the solvent molecules. In some embodiments, thetransformation of crystal modification 2 to crystal modification 1proceeds via a crystalline intermediate, namely modification 12 (seeFIG. 5). In some embodiments, the solvent molecules are removed frommodification 2 without dissolution and recrystallization of thecrystals.

In another aspect, the invention relates to the use of crystalmodification 2 (2A, 2B or 2C) of odevixibat as described herein in aprocess for the preparation of crystal modification 1 of odevixibat.

In yet another aspect, the invention relates to a process for thepreparation of crystal modification 1 of odevixibat. In someembodiments, this process involves isolating crystal modification 2 ofodevixibat from a solution of odevixibat in a solvent mixture comprisingwater and an organic solvent selected from the group consisting ofmethanol, ethanol, 2-propanol, acetone, acetonitrile, 1,4-dioxane, DMFand DMSO, and mixtures thereof. In some embodiments, the processinvolves isolating crystal modification 2 of odevixibat from a solutionof odevixibat in a solvent mixture comprising water and an organicsolvent selected from the group consisting of methanol, ethanol,2-propanol, acetone, acetonitrile, 1,4-dioxane, DMF and DMSO.

In some embodiments, the crystallinity of crystal modification 1 isdependent on the drying process. As is shown in the experimentalsection, it has been observed that superior crystallinity of crystalmodification 1 can be obtained when crystal modification 2 is driedunder vacuum (e.g., less than 5 mbar) or under a nitrogen flow. It isbelieved that drying of crystal modification 2 under these conditionsresults in a dehydrated form, which then quickly takes up water from theair.

In some embodiments, therefore, the process for the preparation ofcrystal modification 1 of odevixibat comprises the steps of:

-   a) isolating crystal modification 2 of odevixibat from a solution of    odevixibat in a solvent mixture comprising water and an organic    solvent selected from the group consisting of methanol, ethanol,    2-propanol, acetone, acetonitrile, 1,4-dioxane, DMF and DMSO; and-   b) drying the solid under vacuum or under a nitrogen flow.

In a preferred embodiment, crystal modification 2 of odevixibat iscrystal modification 2A of odevixibat. In a more preferred embodiment,crystal modification 2A of odevixibat is obtained from a mixture ofwater and ethanol.

In some embodiments, the process for the preparation of crystalmodification 1 of odevixibat comprises the steps of:

-   a) isolating crystal modification 2A of odevixibat from a solution    of odevixibat in a mixture of water and ethanol; and-   b) drying the solid under vacuum or under a nitrogen flow.

In some embodiments, the crystallinity of crystal modification 1 isdependent on the composition of the mixture of water and the organicsolvent. For example, superior crystallinity of crystal modification 1can be obtained from samples of crystal modification 2A that areobtained from a slurry of odevixibat in a 60:40 (% v/v) mixture ofethanol and water at 22° C. In a preferred embodiment, the ethanolcontent in the solvent mixture is about 55 to about 75% (v/v), such asabout 60 to about 70% (v/v). In some embodiments, the ethanol content inthe solvent mixture is about 60% (v/v). In some embodiments, the ethanolcontent in the solvent mixture is about 65% (v/v). In some embodiments,the ethanol content in the solvent mixture is about 70% (v/v).

In some embodiments, the crystallinity of crystal modification 2A isincreased when the isolated crystals are exposed to an ethanol/wateratmosphere containing 40 to 60% (v/v) ethanol for a period of at least24 hours.

In some embodiments, the process comprises the steps of:

-   a) preparing a saturated solution of odevixibat in a mixture of    water and an organic solvent selected from the group consisting of    methanol, ethanol, 2-propanol, acetone, acetonitrile, 1,4-dioxane,    DMF and DMSO;-   b) adding an excess of odevixibat to the saturated solution of    step a) so as to obtain a slurry;-   c) maintaining stirring of the slurry at a temperature of about 0 to    about 25° C., for a period of at least 24 hours;-   d) recovering the solid obtained in step c);-   e) drying the solid under vacuum or under a nitrogen flow.

In some embodiments, the process comprises the steps of:

-   a) preparing a saturated solution of odevixibat in a mixture of    water and ethanol;-   b) adding an excess of odevixibat to the saturated solution of    step a) so as to obtain a slurry;-   c) maintaining stirring of the slurry at a temperature of about 20    to about 25° C., preferably about 22° C., for a period of at least    24 hours;-   d) recovering the solid obtained in step c);-   e) optionally exposing the crystals of step d) to an ethanol/water    atmosphere; and-   f) drying the solid under vacuum or under a nitrogen flow.

Alternatively, crystal modification 1 can be obtained by adding seedcrystals to a saturated solution of odevixibat in a mixture of water anda suitable organic solvent. Thus, in another embodiment, the processcomprises the steps of:

-   a) preparing a saturated solution of odevixibat in a mixture of    water and an organic solvent selected from the group consisting of    methanol, ethanol, 2-propanol, acetone, acetonitrile, 1,4-dioxane,    DMF and DMSO;-   b) adding seed crystals to the saturated solution of step a);-   c) maintaining stirring of the slurry at a temperature of about 0 to    about 25° C., for a period of at least 24 hours;-   d) recovering the solid obtained in step c);-   e) drying the solid under vacuum or under a nitrogen flow.

In some embodiments, the process comprises the steps of:

-   a) preparing a saturated solution of odevixibat in a mixture of    water and ethanol;-   b) adding seed crystals to the saturated solution of step a);-   c) maintaining stirring of the slurry at a temperature of about 20    to about 25° C., preferably 22° C., for a period of at least 24    hours;-   d) recovering the solid obtained in step c);-   e) optionally exposing the crystals of step d) to an ethanol/water    atmosphere; and-   f) drying the solid under vacuum or under a nitrogen flow.

A slurry sample of crystal modification 2 may be used as the seedcrystals. Alternatively, crystal modification 1 may be used. It isbelieved that this form quickly transforms into crystal modification 2when added to the solvent mixture of the crystallization process.

In a further aspect, the invention relates to crystalline modification 1of odevixibat, prepared by a process comprising the steps of:

-   a) isolating crystal modification 2 of odevixibat from a solution of    odevixibat in a solvent mixture comprising water and an organic    solvent selected from the group consisting of methanol, ethanol,    2-propanol, acetone, acetonitrile, 1,4-dioxane, DMF and DMSO; and-   b) drying the solid under vacuum or under a nitrogen flow.

In a further aspect, the invention also relates to crystal modification1 of odevixibat as described herein for use in therapy.

Odevixibat is an ileal bile acid transporter (IBAT) inhibitor. The ilealbile acid transporter (IBAT) is the main mechanism for re-absorption ofbile acids from the GI tract. Partial or full blockade of thatodevixibat mechanism will result in lower concentration of bile acids inthe small bowel wall, portal vein, liver parenchyma, intrahepaticbiliary tree, and extrahepatic biliary tree, including the gall bladder.Diseases which may benefit from partial or full blockade of the IBATmechanism may be those having, as a primary pathophysiological defect,symptoms of excessive concentration of bile acids in serum and in theabove organs. Crystal modification 1 of odevixibat, as described herein,is therefore useful in the treatment or prevention of conditions,disorders and diseases wherein inhibition of the bile acid circulationis desirable, such as cardiovascular diseases, fatty acid metabolism andglucose utilization disorders, gastrointestinal diseases and disorders,liver diseases and disorders.

Cardiovascular diseases and disorders of fatty acid metabolism andglucose utilization include, but are not limited to,hypercholesterolemia; disorders of fatty acid metabolism; type 1 andtype 2 diabetes mellitus; complications of diabetes, includingcataracts, micro- and macrovascular diseases, retinopathy, neuropathy,nephropathy and delayed wound healing, tissue ischaemia, diabetic foot,arteriosclerosis, myocardial infarction, acute coronary syndrome,unstable angina pectoris, stable angina pectoris, stroke, peripheralarterial occlusive disease, cardiomyopathy, heart failure, heart rhythmdisorders and vascular restenosis; diabetes-related diseases such asinsulin resistance (impaired glucose homeostasis), hyperglycemia,hyperinsulinemia, elevated blood levels of fatty acids or glycerol,obesity, dyslipidemia, hyperlipidemia including hypertriglyceridemia,metabolic syndrome (syndrome X), atherosclerosis and hypertension; andfor increasing high density lipoprotein levels.

Gastrointestinal diseases and disorders include constipation (includingchronic constipation, functional constipation, chronic idiopathicconstipation (CIC), intermittent/sporadic constipation, constipationsecondary to diabetes mellitus, constipation secondary to stroke,constipation secondary to chronic kidney disease, constipation secondaryto multiple sclerosis, constipation secondary to Parkinson's disease,constipation secondary to systemic sclerosis, drug induced constipation,irritable bowel syndrome with constipation (IBS-C), irritable bowelsyndrome mixed (IBS-M), pediatric functional constipation and opioidinduced constipation); Crohn's disease; primary bile acid malabsorption;irritable bowel syndrome (IBS); inflammatory bowel disease (IBD); ilealinflammation; and reflux disease and complications thereof, such asBarrett's esophagus, bile reflux esophagitis and bile reflux gastritis.The treatment and prevention of constipation has also been disclosed inWO 2004/089350, which is incorporated by reference in its entiretyherein.

A liver disease as defined herein is any disease in the liver and inorgans connected therewith, such as the pancreas, portal vein, the liverparenchyma, the intrahepatic biliary tree, the extrahepatic biliarytree, and the gall bladder. In some embodiments, a liver disease a bileacid-dependent liver disease. In some embodiments, a liver diseaseinvolves elevated levels of bile acids in the serum and/or in the liver.In some embodiments, a liver disease is a cholestatic liver disease.Liver diseases and disorders include, but are not limited to aninherited metabolic disorder of the liver; inborn errors of bile acidsynthesis; congenital bile duct anomalies; biliary atresia; post-Kasaibiliary atresia; post-liver transplantation biliary atresia; neonatalhepatitis; neonatal cholestasis; hereditary forms of cholestasis;cerebrotendinous xanthomatosis; a secondary defect of BA synthesis;Zellweger's syndrome; cystic fibrosis-associated liver disease;alpha1-antitrypsin deficiency; Alagilles syndrome (ALGS); Bylersyndrome; a primary defect of bile acid (BA) synthesis; progressivefamilial intrahepatic cholestasis (PFIC) including PFIC-1, PFIC-2,PFIC-3 and non-specified PFIC, post-biliary diversion PFIC andpost-liver transplant PFIC; benign recurrent intrahepatic cholestasis(BRIC) including BRIC1, BRIC2 and non-specified BRIC, post-biliarydiversion BRIC and post-liver transplant BRIC; autoimmune hepatitis;primary biliary cirrhosis (PBC); liver fibrosis; non-alcoholic fattyliver disease (NAFLD); non-alcoholic steatohepatitis (NASH); portalhypertension; cholestasis; Down syndrome cholestasis; drug-inducedcholestasis; intrahepatic cholestasis of pregnancy (jaundice duringpregnancy); intrahepatic cholestasis; extrahepatic cholestasis;parenteral nutrition associated cholestasis (PNAC); lowphospholipid-associated cholestasis; lymphedema cholestasis syndrome 1(LSC1); primary sclerosing cholangitis (PSC); immunoglobulin G4associated cholangitis; primary biliary cholangitis; cholelithiasis(gall stones); biliary lithiasis; choledocholithiasis; gallstonepancreatitis; Caroli disease; malignancy of bile ducts; malignancycausing obstruction of the biliary tree; biliary strictures; AIDScholangiopathy; ischemic cholangiopathy; pruritus due to cholestasis orjaundice; pancreatitis; chronic autoimmune liver disease leading toprogressive cholestasis; hepatic steatosis; alcoholic hepatitis; acutefatty liver; fatty liver of pregnancy; drug-induced hepatitis; ironoverload disorders; congenital bile acid synthesis defect type 1 (BAStype 1); drug-induced liver injury (DILI); hepatic fibrosis; congenitalhepatic fibrosis; hepatic cirrhosis; Langerhans cell histiocytosis(LCH); neonatal ichthyosis sclerosing cholangitis (NISCH);erythropoietic protoporphyria (EPP); idiopathic adulthood ductopenia(IAD); idiopathic neonatal hepatitis (INH); non syndromic paucity ofinterlobular bile ducts (NS PILBD); North American Indian childhoodcirrhosis (NAIC); hepatic sarcoidosis; amyloidosis; necrotizingenterocolitis; serum bile acid-caused toxicities, including cardiacrhythm disturbances (e.g., atrial fibrillation) in setting of abnormalserum bile acid profile, cardiomyopathy associated with liver cirrhosis(“cholecardia”), and skeletal muscle wasting associated with cholestaticliver disease; viral hepatitis (including hepatitis A, hepatitis B,hepatitis C, hepatitis D and hepatitis E); hepatocellular carcinoma(hepatoma); cholangiocarcinoma; bile acid-related gastrointestinalcancers; and cholestasis caused by tumours and neoplasms of the liver,of the biliary tract and of the pancreas. The treatment and preventionof liver diseases has also been disclosed in WO 2012/064266, which isincorporated by reference in its entirety herein.

Other diseases that may be treated or prevented by crystal modification1 of odevixibat include hyperabsorption syndromes (includingabetalipoproteinemia, familial hypobetalipoproteinemia (FHBL),chylomicron retention disease (CRD) and sitosterolemia);hypervitaminosis and osteopetrosis; hypertension; glomerularhyperfiltration; and pruritus of renal failure.

Biliary atresia is a rare pediatric liver disease that involves apartial or total blockage (or even absence) of large bile ducts. Thisblockage or absence causes cholestasis that leads to the accumulation ofbile acids that damages the liver. In some embodiments, the accumulationof bile acids occurs in the extrahepatic biliary tree. In someembodiments, the accumulation of bile acids occurs in the intrahepaticbiliary tree. The current standard of care is the Kasai procedure, whichis a surgery that removes the blocked bile ducts and directly connects aportion of the small intestine to the liver. There are currently noapproved drug therapies for this disorder.

Provided herein are methods for treating biliary atresia in a subject inneed thereof, the methods comprising administration of a therapeuticallyeffective amount of crystal modification I of odevixibat. In someembodiments, the subject has undergone the Kasai procedure prior toadministration of a crystal modification I of odevixibat. In someembodiments, the subject is administered crystal modification I ofodevixibat prior to undergoing the Kasai procedure. In some embodiments,the treatment of biliary atresia decreases the level of serum bile acidsin the subject.

In some embodiments, the level of serum bile acids is determined by, forexample, an ELISA enzymatic assay or the assays for the measurement oftotal bile acids as described in Danese et al., PLoS One. 2017, vol.12(6): e0179200, which is incorporated by reference herein in itsentirety. In some embodiments, the level of serum bile acids candecrease by, for example, 10% to 40%, 20% to 50%, 30% to 60%, 40% to70%, 50% to 80%, or by more than 90% of the level of serum bile acidsprior to administration of crystal modification I of odevixibat. In someembodiments, the treatment of biliary atresia includes treatment ofpruritus.

PFIC is a rare genetic disorder that is estimated to affect between onein every 50,000 to 100,000 children born worldwide and causesprogressive, life-threatening liver disease.

One manifestation of PFIC is pruritus, which often results in a severelydiminished quality of life. In some cases, PFIC leads to cirrhosis andliver failure. Current therapies include Partial External BiliaryDiversion (PEBD) and liver transplantation, however, these options cancarry substantial risk of post-surgical complications, as well aspsychological and social issues.

Three alternative gene defects have been identified that correlate tothree separate PFIC subtypes known as types 1, 2 and 3.

-   -   PFIC, type 1, which is sometimes referred to as “Byler disease,”        is caused by impaired bile secretion due to mutations in the        ATP8B1 gene, which codes for a protein that helps to maintain an        appropriate balance of fats known as phospholipids in cell        membranes in the bile ducts. An imbalance in these phospholipids        is associated with cholestasis and elevated bile acids in the        liver. Subjects affected by PFIC, type 1 usually develop        cholestasis in the first months of life and, in the absence of        surgical treatment, progress to cirrhosis and end-stage liver        disease before the end of the first decade of life.    -   PFIC, type 2, which is sometimes referred to as “Byler        syndrome,” is caused by impaired bile salt secretion due to        mutations in the ABCB11 gene, which codes for a protein, known        as the bile salt export pump, that moves bile acids out of the        liver. Subjects with PFIC, type 2 often develop liver failure        within the first few years of life and are at increased risk of        developing a type of liver cancer known as hepatocellular        carcinoma.    -   PFIC, type 3, which typically presents in the first years of        childhood with progressive cholestasis, is caused by mutations        in the ABCB4 gene, which codes for a transporter that moves        phospholipids across cell membranes.

In addition, TJP2 gene, NR1H4 gene or Myo5b gene mutations have beenproposed to be causes of PFIC. In addition, some subjects with PFIC donot have a mutation in any of the ATP8B1, ABCB11, ABCB4, TJP2, NR1H4 orMyo5b genes. In these cases, the cause of the condition is unknown.

Exemplary mutations of the ATP8B1 gene or the resulting protein arelisted in Tables 1 and 2, with numbering based on the human wild typeATP8B1 protein (e.g., SEQ ID NO: 1) or gene (e.g., SEQ ID NO: 2).Exemplary mutations of the ABCB11 gene or the resulting protein arelisted in Tables 3 and 4, with numbering based on the human wild typeABCB11 protein (e.g., SEQ ID NO: 3) or gene (e.g., SEQ ID NO: 4).

As can be appreciated by those skilled in the art, an amino acidposition in a reference protein sequence that corresponds to a specificamino acid position in SEQ ID NO: 1 or 3 can be determined by aligningthe reference protein sequence with SEQ ID NO: 1 or 3 (e.g., using asoftware program, such as ClustalW2). Changes to these residues(referred to herein as “mutations”) may include single or multiple aminoacid substitutions, insertions within or flanking the sequences, anddeletions within or flanking the sequences. As can be appreciated bythose skilled in the art, an nucleotide position in a reference genesequence that corresponds to a specific nucleotide position in SEQ IDNO: 2 or 4 can be determined by aligning the reference gene sequencewith SEQ ID NO: 2 or 4 (e.g., using a software program, such asClustalW2). Changes to these residues (referred to herein as“mutations”) may include single or multiple nucleotide substitutions,insertions within or flanking the sequences, and deletions within orflanking the sequences. See also Kooistra, et al., “KLIFS: A structuralkinase-ligand interaction database,” Nucleic Acids Res. 2016, vol. 44,no. D1, pp. D365-D371, which is incorporated by reference in itsentirety herein.

TABLE 1 Exemplary ATP8B1 Mutations Amino acid position 3 (e.g., T3K)²⁷Amino acid position 23 (e.g., P23L)⁵ Amino acid position 45 (e.g.,N45T)^(5,8,9) Amino acid position 46 (e.g., R46X)^(A,25) Amino acidposition 62 (e.g., C62R)²⁸ Amino acid position 63 (e.g., T63T)⁴¹ Aminoacid position 70 (e.g., D70N)^(1,6) Amino acid position 71 (e.g.,R71H)⁴³ Amino acid position 78 (e.g., H78Q)¹⁹ Amino acid position 82(e.g., T82T)⁴¹ Amino acid position 92 (e.g., Y92Y)⁴¹ Amino acid position93 (e.g., A93A)⁶ Amino acid position 96 (e.g., A96G)²⁷ Amino acidposition 114 (e.g., E114Q)⁸ Amino acid position 127 (e.g., L127P⁶,L127V³⁶) Amino acid position 177 (e.g., T177T)⁶ Amino acid position 179(e.g., E179X)²⁹ Δ Amino acid positions 185-282⁴⁴ Amino acid position 197(e.g., G197Lfs*10)²² Amino acid position 201 (e.g., R201S²⁷, R201H³⁵)Amino acid position 203 (e.g., K203E^(5,8), K203R⁹, K203fs²⁵) Amino acidposition 205 (e.g., N205fs⁶, N205Kfs*2³⁵) Amino acid position 209 (e.g.,P209T)⁴ Amino acid position 217 (e.g., S217N)⁴³ Amino acid position 232(e.g., D232D)³⁰ Amino acid position 233 (e.g., G233R)³⁸ Amino acidposition 243 (e.g., L243fs*28)³³ Amino acid position 265 (e.g., C265R)²⁵Amino acid position 271 (e.g., R271X¹³, R271R³⁰) Amino acid position 288(e.g., L288S)⁶ Amino acid position 294 (e.g., L294S)⁴³ Amino acidposition 296 (e.g., R296C)¹¹ Amino acid position 305 (e.g., F305I)²⁸Amino acid position 306 (e.g., C306R)²³ Amino acid position 307 (e.g.,H307L)³⁵ Amino acid position 308 (e.g., G308V¹, G308D⁶, G308S³⁵) Aminoacid position 314 (e.g., G314S)¹³ Amino acid position 320 (e.g.,M320Vfs*13)¹¹ Amino acid position 337 (e.g., M337R)¹⁸ Amino acidposition 338 (e.g., N338K)¹⁸ Amino acid position 340 (e.g., M340V)¹⁸Amino acid position 344 (e.g., I344F)^(6,20) Amino acid position 349(e.g., I349T)⁴¹ Amino acid position 358 (e.g., G358R)²⁸ Amino acidposition 367 (e.g., G367G)⁴¹ Amino acid position 368 (e.g., N368D)⁴¹Amino acid position 393 (e.g., I393V)²⁷ Amino acid position 403 (e.g.,S403Y)⁶ Amino acid position 407 (e.g., S407N)⁴⁰ Amino acid position 412(e.g., R412P)⁶ Amino acid position 415 (e.g., Q415R)²⁷ Amino acidposition 422 (e.g., D422H)³⁵ Amino acid position 429 (e.g., E429A)⁶Amino acid position 446 (e.g., G446R)^(4,11) Amino acid position 453(e.g., S453Y)⁶ Amino acid position 454 (e.g., D454G)⁶ Amino acidposition 455 (e.g., K455N)⁴³ Amino acid position 456 (e.g., T456M^(3,6),T456K³⁵) Amino acid position 457 (e.g., G457G⁶, G457fs*6³³) Amino acidposition 469 (e.g., C469G)⁴¹ Amino acid position 478 (e.g., H478H)⁴¹Amino acid position 500 (e.g., Y500H)⁶ Amino acid position 525 (e.g.,R525X)⁴ Δ Amino acid position 529⁶ Amino acid position 535 (e.g.,H535L⁶, H535N⁴¹) Amino acid position 553 (e.g., P553P)⁴³ Amino acidposition 554 (e.g., D554N^(1,6), D554A³⁵) Δ Amino acid positions556-628⁴⁴ Δ Amino acid positions 559-563³⁵ Amino acid position 570(e.g., L570L)⁴¹ Amino acid position 577 (e.g., I577V)¹⁹ Amino acidposition 581 (e.g., E581K)³⁵ Amino acid positions 554 and 581 (e.g.,D554A + E581K)³⁵ Amino acid position 585 (e.g., E585X)²¹ Amino acidposition 600 (e.g., R600W^(2,4), R600Q⁶) Amino acid position 602 (e.g.,R602X)^(3,6) Amino acid position 628 (e.g., R628W)⁶ Amino acid position631 (e.g., R631Q)²⁸ Δ Amino acid positions 645-699⁴ Amino acid position661 (e.g., I661T)^(1,4,6) Amino acid position 665 (e.g., E665X)^(4,6)Amino acid position 672 (e.g., K672fs⁶, K672Vfs*1³⁵) Amino acid position674 (e.g., M674T)¹⁹ Amino acid positions 78 and 674 (e.g., H78Q/M674T)¹⁹Amino acid position 684 (e.g., D684D)⁴¹ Amino acid position 688 (e.g.,D688G)⁶ Amino acid position 694 (e.g., I694T⁶, I694N¹⁷) Amino acidposition 695 (e.g., E695K)²⁷ Amino acid position 709 (e.g., K709fs⁶,K709Qfs*41¹³) Amino acid position 717 (e.g., T717N)⁴ Amino acid position733 (e.g., G733R)⁶ Amino acid position 757 (e.g., Y757X)⁴ Amino acidposition 749 (e.g., L749P)²¹ Amino acid position 792 (e.g., P792fs)⁶ ΔAmino acid position 795-797⁶ Amino acid position 809 (e.g., I809L)²⁷Amino acid position 814 (e.g., K814N)²⁸ Amino acid position 833 (e.g.,R833Q²⁷, R833W⁴¹) Amino acid position 835 (e.g., K835Rfs*36)³⁵ Aminoacid position 845 (e.g., K845fs)²⁵ Amino acid position 849 (e.g.,R849Q)²⁴ Amino acid position 853 (e.g., F853S, F853fs)⁶ Amino acidposition 867 (e.g., R867C¹, R867fs⁶, R867H²³) Amino acid position 885(e.g., K885T)⁴¹ Amino acid position 888 (e.g., T888T)⁴¹ Amino acidposition 892 (e.g., G892R)⁶ Amino acid position 912 (e.g., G912R)³⁵Amino acid position 921 (e.g., S921S)⁴¹ Amino acid position 924 (e.g.,Y924C)²⁸ Amino acid position 930 (e.g., R930X⁶, R930Q²⁸) Amino acidposition 941 (e.g., R941X)³⁵ Amino acid position 946 (e.g., R946T)⁴¹Amino acid position 952 (e.g., R952Q^(5,9,15), R952X⁶) Amino acidposition 958 (e.g., N958fs)⁶ Amino acid position 960 (e.g., A960A)⁴¹ ΔAmino acid position 971⁴³ Amino acid position 976 (e.g., A976E⁴¹,A976A⁴³) Amino acid position 981 (e.g., E981K)²⁰ Amino acid position 994(e.g., S994R)⁴ Amino acid position 1011 (e.g., L1011fs*18)³³ Amino acidposition 1012 (e.g., S1012I)¹⁰ Amino acid position 1014 (e.g.,R1014X)^(6,11) Amino acid position 1015 (e.g., F1015L)²⁷ Amino acidposition 1023 (e.g., Q1023fs)⁶ Amino acid position 1040 (e.g.,G1040R)^(1,6) Amino acid position 1044 (e.g., S0144L)³⁴ Amino acidposition 1047 (e.g., L1047fs)⁶ Amino acid position 1050 (e.g., I1050K)³¹Amino acid position 1052 (e.g., L1052R)²⁸ Amino acid position 1095(e.g., W1095X)¹¹ Amino acid position 1098 (e.g., V1098X)³⁵ Amino acidposition 1131 (e.g., Q1131X)⁴⁴ Amino acid position 1142 (e.g.,A1142Tfs*35)⁴³ Amino acid position 1144 (e.g., Y1144Y)⁴³ Amino acidposition 1150 (e.g., I1150T)⁴¹ Amino acid position 1152 (e.g., A1152T)³⁰Amino acid position 1159 (e.g., P1159P)^(25,43) Amino acid position 1164(e.g., R1164X)⁶ Amino acid position 1193 (e.g., R1193fs*39)³³ Amino acidposition 1197 (e.g., V1197L)⁴¹ Amino acid position 1208 (e.g., A1208fs)⁶Amino acid position 1209 (e.g., Y1209Lfs*28)⁴ Amino acid position 1211(e.g., F1211L)²⁷ Amino acid position 1219 (e.g., D1219H⁵, D1219G²⁷)Amino acid position 1223 (e.g., S1223S)⁴¹ Amino acid position 1233(e.g., P1233P)⁴¹ Amino acid position 1241 (e.g., G1241fs)⁶ Amino acidposition 1248 (e.g., T1248T)⁴³ Splice site mutation IVS3 + 1_+3delGTG⁶Splice site mutation IVS3 − 2A > G⁶ IVS6 + 5T > G^(17,25) Splice sitemutation IVS8 + 1G > T⁶ IVS9 − G > A²⁶ IVS12 + 1G > A²⁵ Splice sitemutation IVS17 − 1G > A⁶ Splice site mutation IVS18 + 2T > C⁶ Splicesite mutation IVS20 − 4CT > AA Splice site mutation IVS21 + 5G > A⁶Splice site mutation IVS23 − 3C > A⁶ Splice site mutation IVS26 + 2T >A⁶ g.24774-42062del⁴ c.-4C > G⁴¹ c.145C > T¹² c.181 − 72G > A⁹ c.182 −5T > A⁴¹ c.182 − 72G > A⁴¹ c.246A > G⁹ c.239G > A³⁹ c.279 + 1_279 +3delGTG⁴⁶ c.280 − 2A > G⁴⁶ c.625_62715delinsACAGTAAT⁴⁶ c.554 + 122C > T⁹c.555 − 3T > C²⁷ c.625 + 5 G > T⁴ Amino acid position 209 (e.g., P209T)and c.625 + 5 G > T⁴ c.628 − 30G > A⁴¹ c.628 − 31C > T⁴¹ c.698 + 1G >T⁴⁶ c.698 + 20C > T⁴¹ c.782 − 1G > A⁴⁶ c.782 − 34G > A⁴¹ Δ795-797¹⁴c.782 − 1G > A⁴ c.852A > C²⁷ c.941 − 1G > A⁴⁶ c.1014C > T⁹ c.1029 +35G > A⁹ c.1221-8C.G⁴¹ 1226delA¹⁶ c.1429 + 1G > A⁴⁶ c.1429 + 2T > G¹³c.1429 + 49G > A⁴¹ c.1430 − 42A > G⁴¹ c.1493T > C¹² c.1587_1589delCTT⁴⁶c.1630 + 2T > G²⁷ c.1631 − 10T > A⁴¹ c.1637 − 37T > C⁴¹ 1660 G > A¹⁴1798 C > T¹⁴ 1799 G > A¹⁴ c.1819 − 39_41delAA⁹ c.1819 + 1G > A³¹ c.1820− 27G > A⁴¹ c.1918 + 8C > T²⁷ c.1933 − 1G > AK46 c.2097 + 2T > C³²c.2097 + 60T > G⁴¹ c.2097 + 89T > C⁴¹ c.2097 + 97T > G⁴¹ c.2210 − 114T >C⁹ 2210delA¹⁶ c.2210 − 45_50dupATAAAA⁹ c.2285 + 29C · T⁴¹ c.2285 + 32A >G⁴¹ c.2286 − 4_2286-3delinsAA⁴⁶ c.2418 + 5G > A⁴⁶ c.2707 + 3G > C²⁷c.2707 + 9T > G⁴¹ c.2707 + 43A > G⁴¹ c.2709 − 59T > C⁴¹ c.2931 + 9A >G⁴¹ c.2931 + 59T > A⁴¹ C.2932 − 3C > A⁴⁶ c.2932 + 59T > A⁹ c.2937A > C²⁷c.3016 − 9C > A³¹ c.3033-3034del¹⁹3122delTCCTA/insACATCGATGTTGATGTTAGG⁴⁵ 3318 G > A¹⁴ c.3400 + 2T > A⁴⁶c.3401 − 175C > T⁹ c.3401 − 167C > T⁹ c.3401 − 108C > T⁹ c.3531 + 8G >T^(9,15) c.3532 − 15C > T⁹ Δ Phe ex 15⁴ Ex1_Ex13del⁶ Ex2_Ex6del³³Ex12_Ex14del²⁷ Skipped Exon 24⁴⁵ del5′UTR-ex18¹¹ c.*11C > T⁴¹ c.*1101 +366G > A⁷ g.92918del565³¹ GC preceding exon 16 (e.g., resulting in a 4bp deletion)⁴² Frameshift from the 5′ end of exon 16⁴² 5′ 1.4 kbdeletion⁴⁶

TABLE 2 Selected ATP8B1 Mutations Associated with PFIC-1 Amino acidposition 23 (e.g., P23L)⁵ Amino acid position 78 (e.g., H78Q)¹⁹ Aminoacid position 93 (e.g., A93A)⁶ Amino acid position 96 (e.g., A96G)²⁷Amino acid position 127 (e.g., L127P)⁶ Amino acid position 197 (e.g.,G197Lfs*10)²² Amino acid position 205 (e.g., N205fs)⁶ Amino acidposition 209 (e.g., P209T)⁴ Amino acid position 233 (e.g., G233R)³⁸Amino acid position 243 (e.g., L243fs*28)³³ Amino acid position 288(e.g., L288S)⁶ Amino acid position 296 (e.g., R296C)¹¹ Amino acidposition 308 (e.g., G308V^(1,6)) Amino acid position 320 (e.g.,M320Vfs*13)¹¹ Amino acid position 403 (e.g., S403Y)⁶ Amino acid position407 (e.g., S407N)⁴⁰ Amino acid position 412 (e.g., R412P)⁶ Amino acidposition 415 (e.g., Q415R)²⁷ Amino acid position 429 (e.g., E429A)⁶Amino acid position 446 (e.g., G446R)⁴ Amino acid position 456 (e.g.,T456M)^(3,6) Amino acid position 457 (e.g., G457G⁶, G457fs*6³³) Aminoacid position 500 (e.g., Y500H)⁶ Amino acid position 525 (e.g., R525X)⁴Δ Amino acid position 529⁶ Amino acid position 535 (e.g., H535L)⁶ Aminoacid position 554 (e.g., D554N)^(1,6) Amino acid position 577 (e.g.,I577V)¹⁹ Amino acid position 585 (e.g., E585X)²¹ Amino acid position 600(e.g., R600W)⁴ Amino acid position 602 (e.g., R602X)^(3,6) Amino acidposition 661 (e.g., I661T)^(4,6) Amino acid position 665 (e.g.,E665X)^(4,6) Δ Amino acid positions 645-699⁴ Amino acid position 672(e.g., K672fs)⁶ Amino acid position 674 (e.g., M674T)¹⁹ Amino acidpositions 78 and 674 (e.g., H78Q/M674T)¹⁹ Amino acid position 688 (e.g.,D688G)⁶ Amino acid position 694 (e.g., I694N)¹⁷ Amino acid position 695(e.g., E695K)²⁷ Amino acid position 709 (e.g., K709fs)⁶ Amino acidposition 717 (e.g., T717N)⁴ Amino acid position 733 (e.g., G733R)⁶ Aminoacid position 749 (e.g., L749P)²¹ Amino acid position 757 (e.g., Y757X)⁴Amino acid position 792 (e.g., P792fs)⁶ Amino acid position 809 (e.g.,I809L)²⁷ Amino acid position 853 (e.g., F853S, F853fs)⁶ Amino acidposition 867 (e.g., R867fs)⁶ Amino acid position 892 (e.g., G892R)⁶Amino acid position 930 (e.g., R930X⁶, R952Q¹⁵) Amino acid position 952(e.g., R952X)⁶ Amino acid position 958 (e.g., N958fs)⁶ Amino acidposition 981 (e.g., E981K)²⁰ Amino acid position 994 (e.g., S994R)⁴Amino acid position 1014 (e.g., R1014X)^(6,11) Amino acid position 1015(e.g., F1015L)²⁷ Amino acid position 1023 (e.g., Q1023fs)⁶ Amino acidposition 1040 (e.g., G1040R)^(1,6) Amino acid position 1047 (e.g.,L1047fs)⁶ Amino acid position 1095 (e.g., W1095X)¹¹ Amino acid position1208 (e.g., A1208fs)⁶ Amino acid position 1209 (e.g., Y1209Lfs*28)⁴Amino acid position 1211 (e.g., F1211L)²⁷ Amino acid position 1219(e.g., D1219H⁵, D1219G²⁷) Splice site mutation IVS3 + 1_+3delGTG⁶ Splicesite mutation IVS3 − 2A > G⁶ IVS6 + 5T > G¹⁷ Splice site mutation IVS8 +1G > T⁶ IVS9 − G > A²⁶ Splice site mutation IVS17 − 1G > A⁶ Splice sitemutation IVS18 + 2T > C⁶ Splice site mutation IVS21 + 5G > A⁶g.24774-42062del⁴ c.145C > T¹² c.239G > A³⁹ c.625 + 5 G > T⁴ Amino acidposition 209 (e.g., P209T) and c.625 + 5 G > T⁴ c.782 − 1G > A⁴c.1493T > C¹² c.1630 + 2T > G²⁷ 1660 G > A¹⁴ c.2707 + 3G > C²⁷ c.2097 +2T > C³² c.3033-3034del¹⁹ 3318 G > A¹⁴ c.3158 + 8G > T¹⁵ Δ Phe ex 15⁴Ex1_Ex13del⁶ Ex2_Ex6del³³ Ex12_Ex14del²⁷ del5′UTR-ex18¹¹ c.*1101 +366G > A⁷ GC preceding exon 16 (e.g., resulting in a 4 bp deletion)⁴²Frameshift from the 5′ end of exon 16⁴² ^(A) A mutation to ‘X’ denotesan early stop codon

REFERENCES FOR TABLES 1 AND 2

-   ¹ Folmer et al., Hepatology. 2009, vol. 50(5), p. 1597-1605.-   ² Hsu et al., Hepatol Res. 2009, vol. 39(6), p. 625-631.-   ³ Alvarez et al., Hum Mol Genet. 2004, vol. 13(20), p. 2451-2460.-   ⁴ Davit-Spraul et al., Hepatology 2010, vol. 51(5), p. 1645-1655.-   ⁵ Vitale et al., J Gastroenterol. 2018, vol. 53(8), p. 945-958.-   ⁶ Klomp et al., Hepatology 2004, vol. 40(1), p. 27-38.-   ⁷ Zarenezhad et al., Hepatitis Monthly: 2017, vol. 17(2); e43500.-   ⁸ Dixon et al., Scientific Reports 2017, vol. 7, 11823.-   ⁹ Painter et al., Eur J Hum Genet. 2005, vol. 13(4), p. 435-439.-   ¹⁰ Deng et al., World J Gastroenterol. 2012, vol. 18(44), p.    6504-6509.-   ¹¹ Giovannoni et al., PLoS One. 2015, vol. 10(12): e0145021.-   ¹² Li et al., Hepatology International 2017, vol. 11, No. 1, Supp.    Supplement 1, pp. S180. Abstract Number: OP284.-   ¹³ Togawa et al., Journal of Pediatric Gastroenterology and    Nutrition 2018, vol. 67, Supp. Supplement 1, pp. S363. Abstract    Number: 615.-   ¹⁴ Miloh et al., Gastroenterology 2006, vol. 130, No. 4, Suppl. 2,    pp. A759-A760. Meeting Info.: Digestive Disease Week Meeting/107th    Annual Meeting of the American-Gastroenterological-Association. Los    Angeles, Calif., USA. May 19.-   ¹⁵ Dröge et al., Zeitschrift fur Gastroenterologie 2015, vol. 53,    No. 12. Abstract Number: A3-27. Meeting Info: 32. Jahrestagung der    Deutschen Arbeitsgemeinschaft zum Studium der Leber. Dusseldorf,    Germany. 22 Jan. 2016-23 Jan. 2016-   ¹⁶ Mizuochi et al., Clin Chim Acta. 2012, vol. 413(15-16), p.    1301-1304.-   ¹⁷ Liu et al., Hepatology International 2009, vol. 3, No. 1, p.    184-185. Abstract Number: PE405. Meeting Info: 19th Conference of    the Asian Pacific Association for the Study of the Liver. Hong Kong,    China. 13 Feb. 2009-16 Feb. 2009-   ¹⁸ McKay et al., Version 2. F1000Res. 2013; 2: 32. DOI:    10.12688/f1000research.2-32.v2-   ¹⁹ Hasegawa et al., Orphanet J Rare Dis. 2014, vol. 9:89.-   ²⁰ Stone et al., J Biol Chem. 2012, vol. 287(49), p. 41139-51.-   ²¹ Kang et al., J Pathol Transl Med. 2019 May 16. doi:    10.4132/jptm.2019.05.03. [Epub ahead of print]-   ²² Sharma et al., BMC Gastroenterol. 2018, vol. 18(1), p. 107.-   ²³ Uegaki et al., Intern Med. 2008, vol. 47(7), p. 599-602.-   ²⁴ Goldschmidt et al., Hepatol Res. 2016, vol. 46(4), p. 306-311.-   ²⁵ Liu et al., J Pediatr Gastroenterol Nutr. 2010, vol. 50(2), p.    179-183.-   ²⁶ Jung et al., J Pediatr Gastroenterol Nutr. 2007, vol. 44(4), p.    453-458.-   ²⁷ Bounford. University of Birmingham. Dissertation Abstracts    International, (2016) Vol. 75, No. 1C. Order No.: AA110588329.    ProQuest Dissertations & Theses.-   ²⁸ Stolz et al., Aliment Pharmacol Ther. 2019, vol. 49(9), p.    1195-1204.-   ²⁹ Ivashkin et al., Hepatology International 2016, vol. 10, No. 1,    Supp. SUPPL. 1, pp. S461. Abstract Number: LBO-38. Meeting Info:    25th Annual Conference of the Asian Pacific Association for the    Study of the Liver, APASL 2016. Tokyo, Japan. 20 Feb. 2016-24 Feb.    2016-   ³⁰ Blackmore et al., J Clin Exp Hepatol. 2013, vol. 3(2), p.    159-161.-   ³¹ Matte et al., J Pediatr Gastroenterol Nutr. 2010, vol. 51(4), p.    488-493.-   ³² Squires et al., J Pediatr Gastroenterol Nutr. 2017, vol.    64(3), p. 425-430.-   ³³ Hayshi et al., EBioMedicine. 2018, vol. 27, p. 187-199.-   ³⁴ Nagasaka et al., J Pediatr Gastroenterol Nutr. 2007, vol.    45(1), p. 96-105.-   ³⁵ Wang et al., PLoS One. 2016; vol. 11(4): e0153114.-   ³⁶ Narchi et al., Saudi J Gastroenterol. 2017, vol. 23(5), p.    303-305.-   ³⁷ Alashkar et al., Blood 2015, vol. 126, No. 23. Meeting Info.:    57th Annual Meeting of the American-Society-of-Hematology. Orlando,    Fla., USA. Dec. 5-8, 2015. Amer Soc Hematol.-   ³⁸ Ferreira et al., Pediatric Transplantation 2013, vol. 17, Supp.    SUPPL. 1, pp. 99. Abstract Number: 239. Meeting Info: IPTA 7th    Congress on Pediatric Transplantation. Warsaw, Poland. 13 Jul.    2013-16 Jul. 2013.-   ³⁹ Pauli-Magnus et al., J Hepatol. 2005, vol. 43(2), p. 342-357.-   ⁴⁰ Jericho et al., Journal of Pediatric Gastroenterology and    Nutrition 2015, vol. 60(3), p. 368-374.-   ⁴¹ van der Woerd et al., PLoS One. 2013, vol. 8(11): e80553.-   ⁴² Copeland et al., J Gastroenterol Hepatol. 2013, vol. 28(3), p.    560-564.-   ⁴³ Dröge et al., J Hepatol. 2017, vol. 67(6), p. 1253-1264.-   ⁴⁴ Chen et al., Journal of Pediatrics 2002, vol. 140(1), p. 119-124.-   ⁴⁵ Jirsa et al., Hepatol Res. 2004, vol. 30(1), p. 1-3.-   ⁴⁶ van der Woerd et al., Hepatology 2015, vol. 61(4), p. 1382-1391.

In some embodiments, the mutation in ATP8B1 is selected from L127P,G308V, T456M, D554N, F529del, I661T, E665X, R930X, R952X, R1014X, andG1040R.

TABLE 3 Exemplary ABCB11 Mutations Amino acid position 1 (e.g., M1V)⁹Amino acid position 4 (e.g., S4X)^(A,64) Amino acid position 8 (e.g.,R8X)⁸⁸ Amino acid position 19 (e.g., G19R)⁵⁶ Amino acid position 24(e.g., K24X)³⁵ Amino acid position 25 (e.g., S25X)^(5,14) Amino acidposition 26 (e.g., Y26Ifs*7)³⁸ Amino acid position 36 (e.g., D36D)²⁷Amino acid position 38 (e.g., K38Rfs*24)⁷³ Amino acid position 43 (e.g.,V43I)⁵⁷ Amino acid position 49 (e.g., Q49X)⁷³ Amino acid position 50(e.g., L50S, L50W)⁵⁷ Amino acid position 52 (e.g., R52W²⁶, R52R²⁸) Aminoacid position 56 (e.g., S56L)⁵⁸ Amino acid position 58 (e.g., D58N)⁶²Amino acid position 62 (e.g., M62K)⁹ Amino acid position 66 (e.g.,S66N)¹⁷ Amino acid position 68 (e.g., C68Y)⁴¹ Amino acid position 50(e.g., L50S)^(5,7) Amino acid position 71 (e.g., L71H)⁷³ Amino acidposition 74 (e.g., I74R)⁷¹ Amino acid position 77 (e.g., P77A)⁷³ Aminoacid position 87 (e.g., T87R)⁶⁷ Amino acid position 90 (e.g.,F90F)^(7,27) Amino acid position 93 (e.g., Y93S¹³, Y93X⁸⁸) Amino acidposition 96 (e.g., E96X)⁸⁸ Amino acid position 97 (e.g., L97X)³⁹ Aminoacid position 101 (e.g., Q101Dfs*8)⁹ Amino acid position 107 (e.g.,C107R)³⁶ Amino acid position 112 (e.g., I112T)⁹ Amino acid position 114(e.g., W114R)^(2,9) Amino acid position 123 (e.g. M123T)⁶⁷ Amino acidposition 127 (e.g., T127Hfs*6)⁵ Amino acid position 129 (e.g., C129Y)²⁵Amino acid position 130 (e.g., G130G)⁷⁷ Amino acid position 134 (e.g.,I134I)²⁸ Amino acid position 135 (e.g., E135K^(7,13), E135L¹⁷) Aminoacid position 137 (e.g., E137K)⁷ Amino acid position 157 (e.g., Y157C)⁵Amino acid position 161 (e.g., C161X)³⁹ Amino acid position 164 (e.g.,V164Gfs*7³⁰, V164I⁸⁵) Amino acid position 167 (e.g., A167S⁴, A167V⁷,A167T^(9,17)) Amino acid position 181 (e.g., R181I)³⁵ Amino acidposition 182 (e.g., I182K)⁹ Amino acid position 183 (e.g., M183V⁸,M183T⁹) Amino acid position 185 (e.g., M185I)⁷³ Amino acid position 186(e.g., E186G)^(2,7,22) Amino acid position 188 (e.g., G188W)⁷³ Aminoacid position 194 (e.g., S194P)⁷ Amino acid position 198 (e.g., L198P)⁷Amino acid position 199 (e.g., N199Ifs*15X)⁸⁸ Amino acid position 206(e.g., 1206V)²⁸ Amino acid position 212 (e.g., A212T)⁷³ Amino acidposition 217 (e.g., M217R)⁸⁸ Amino acid position 225 (e.g., T225P)⁵⁷Amino acid position 226 (e.g., S226L)⁹ Amino acid position 232 (e.g.,L232Cfs*9)⁹ Amino acid position 233 (e.g., L233S)⁸⁶ Amino acid position238 (e.g., G238V)^(2,7) Amino acid position 242 (e.g., T242I)^(5,7)Amino acid position 245 (e.g., I245Tfs*26)⁵⁷ Amino acid position 256(e.g., A256G)⁹ Amino acid position 260 (e.g., G260D)⁷ Amino acidposition 269 (e.g., Y269Y)²⁷ Amino acid position 277 (e.g., A277E)⁷⁷Amino acid position 283 (e.g., E283D)⁷³ Amino acid positions 212 and 283(e.g., A212T + E283D)⁷³ Amino acid position 284 (e.g., V284L^(7,39),V284A⁷, V284D²³) Amino acid position 297 (e.g., E297G^(1,2,5,7), E297K⁷)Amino acid position 299 (e.g., R299K)²⁸ Amino acid position 303 (e.g.,R303K⁸, R303M⁶³ R303fsX321⁸³) Amino acid position 304 (e.g., Y304X)²⁶Amino acid position 312 (e.g., Q312H)⁷ Amino acid position 313 (e.g.,R313S)^(5,7) Amino acid position 314 (e.g., W314X)⁵⁷ Amino acid position318 (e.g., K318Rfs*26)²⁹ Amino acid position 319 (e.g., G319G)⁷ Aminoacid position 327 (e.g., G327E)^(5,7) Amino acid position 330 (e.g.,W330X)²⁴ Amino acid position 336 (e.g., C336S)^(2,7) Amino acid position337 (e.g., Y337H)^(21,27) Amino acid position 342 (e.g., W342G)⁵⁰ Aminoacid position 354 (e.g., R354X)⁹ Amino acid position 361 (e.g., Q361X⁵⁷,Q361R⁷⁴) Amino acid position 366 (e.g., V366V²⁸, V366D⁵⁷) Amino acidposition 368 (e.g., V368Rfs*27)⁵ Amino acid position 374 (e.g., G374S)³Amino acid position 380 (e.g., L380Wfs*18)⁵ Amino acid position 382(e.g., A382G)⁸⁸ Δ Amino acid positions 382-388⁵ Δ Amino acid positions383-389⁵⁷ Amino acid position 387 (e.g., R387H)⁹ Amino acid position 390(e.g., A390P)^(5,7) Amino acid position 395 (e.g., E395E)²⁸ Amino acidposition 404 (e.g., D404G)⁹ Amino acid position 410 (e.g., G410D)^(5,7)Amino acid position 413 (e.g., L413W)^(5,7) Amino acid position 415(e.g., R415X)⁴² Amino acid position 416 (e.g., I416I)²⁷ Amino acidposition 420 (e.g., I420T)⁹ Amino acid position 423 (e.g., H423R)¹³Amino acid position 432 (e.g., R432T)^(1,2,7) Amino acid position 436(e.g., K436N)⁴⁰ Amino acid position 440 (e.g., D440E)⁸⁸ Amino acidposition 444 (e.g., V444A)² Amino acid position 454 (e.g., V454X)⁴⁹Amino acid position 455 (e.g., G455E)⁹ Amino acid position 457 (e.g.,S457Vfs*23)⁸⁸ Amino acid position 461 (e.g., K461E)^(2,7) Amino acidposition 462 (e.g., S462R)⁸⁸ Amino acid position 463 (e.g., T463I)^(5,7)Amino acid position 466 (e.g., Q466K)^(5,7) Amino acid position 470(e.g., R470Q^(5,7), R470X⁹) Amino acid position 471 (e.g., Y472X)⁵ Aminoacid position 472 (e.g., Y472C^(5,27), Y472X¹⁴) Amino acid position 473(e.g., D473Q³⁵, D473V⁸⁸) Amino acid position 475 (e.g., C475X)²⁹ Aminoacid position 481 (e.g., V481E)^(5,7) Amino acid position 482 (e.g.,D482G)^(2,5,7) Amino acid position 484 (e.g., H484Rfs*5)⁹ Amino acidposition 487 (e.g., R487H², R487P⁵) Amino acid position 490 (e.g.,N490D)^(5,7) Amino acid position 493 (e.g., W493X)⁸ Amino acid positon496 (e.g., D496V)⁸⁸ Amino acid position 498 (e.g., I498T)^(2,7) Aminoacid position 499 (e.g., G499E)⁷³ Amino acid position 501 (e.g.,V501G)⁶⁸ Amino acid position 504 (e.g., E504K)⁷⁹ Amino acid position 510(e.g., T510T)⁷ Amino acid position 512 (e.g., I512T)^(5,7) Amino acidposition 515 (e.g., N515T^(5,7), N515D⁶⁴) Amino acid position 516 (e.g.,I516M)¹⁷ Amino acid position 517 (e.g., R517H)^(5,7) Amino acid position520 (e.g., R520X)⁵ Amino acid position 523 (e.g., A523G)¹³ Amino acidposition 528 (e.g., I528Sfs*21⁵, I528X⁹, I528T⁷³) Amino acid position535 (e.g., A535A⁷, A535X⁸⁹) Amino acid position 540 (e.g., F540L)⁴⁶Amino acid position 541 (e.g., I541L^(5,7), I541T^(5,17)) Amino acidposition 546 (e.g., Q546K³⁹, Q546H⁷³) Amino acid position 548 (e.g.,F548Y)^(5,7) Amino acid position 549 (e.g., D549V)⁹ Amino acid position554 (e.g., E554K)²¹ Amino acid position 556 (e.g., G556R)⁶⁷ Amino acidposition 558 (e.g., Q558H)²³ Amino acid position 559 (e.g., M559T)⁵⁷Amino acid position 562 (e.g., G562D^(5,7), G562S⁷³) Amino acid position570 (e.g., A570T^(2,5,7), A570V²⁶) Amino acid position 575 (e.g.,R575X^(2,5), R575Q²¹) Amino acid position 580 (e.g., L580P)⁵⁷ Amino acidposition 586 (e.g., T586I)⁷ Amino acid position 587 (e.g., S587X)⁷³Amino acid position 588 (e.g., A588V^(5,7), A588P⁷³) Amino acid position591 (e.g., N591S)^(2,7) Amino acid position 593 (e.g., S593R)^(2,7)Amino acid position 597 (e.g., V597V⁹, V597L¹³) Amino acid position 603(e.g., K603K)⁵⁵ Amino acid position 609 (e.g., H609Hfs*46)²⁶ Amino acidposition 610 (e.g., I610Gfs*45⁹, I610T⁵⁷)⁹ Amino acid position 615(e.g., H615R)²⁶ Amino acid position 616 (e.g., R616G²⁸, R616H⁷³) Aminoacid position 619 (e.g., T619A)²⁸ Amino acid position 623 (e.g.,A623A)²⁸ Amino acid position 625 (e.g., T625Nfs*5)²⁶ Amino acid position627 (e.g., I627T)⁷ Amino acid position 628 (e.g., G628Wfs*3)⁷⁰ Aminoacid position 636 (e.g., E636G)² Amino acid position 648 (e.g.,G648Vfs*6⁵, G648V⁵⁰) Amino acid position 655 (e.g., T655I)⁷ Amino acidposition 669 (e.g., I669V)²⁶ Amino acid position 676 (e.g., D676Y)¹¹Amino acid position 677 (e.g., M677V)^(7,13) Amino acid position 679(e.g., A679V)⁵⁸ Amino acid position 685 (e.g., G685W)⁶⁰ Amino acidposition 696 (e.g., R696W²⁷, R696Q⁵⁸) Amino acid position 698 (e.g.,R698H^(7,9), R698K⁶¹, R698C⁸⁸) Amino acid position 699 (e.g., S699P)⁹Amino acid position 701 (e.g., S701P)⁵⁸ Amino acid position 702 (e.g.,Q702X)⁸⁹ Amino acid position 709 (e.g., E709K)⁷ Amino acid position 710(e.g., P710P)⁷ Amino acid position 712 (e.g., L712L)²⁸ Amino acidposition 721 (e.g., Y721C)⁸⁸ Amino acid position 729 (e.g., D724N)³⁹Amino acid position 731 (e.g., P731S)²³ Amino acid position 740 (e.g.,P740Qfs*6)⁷³ Amino acid position 758 (e.g., G758R)⁵ Amino acid position766 (e.g., G766R)^(5,24) Amino acid position 772 (e.g., Y772X)⁵ Aminoacid position 804 (e.g., A804A)⁷ Amino acid position 806 (e.g., G806D⁴⁴,G806G⁵⁵) Amino acid position 809 (e.g., S809F)⁸¹ Amino acid position 817(e.g., G817G)⁸⁸ Amino acid position 818 (e.g., Y818F)⁷ Amino acidposition 824 (e.g., G824E)⁴² Amino acid position 825 (e.g., G825G)⁷³Amino acid position 830 (e.g., R830Gfs*28)⁷³ Amino acid position 832(e.g., R832C^(7,26), R832H⁴¹) Amino acid position 842 (e.g., D842G)²Amino acid position 848 (e.g., D848N)⁷³ Amino acid position 855 (e.g.,G855R)¹¹ Amino acid position 859 (e.g., T859R)^(5,7) Amino acid position865 (e.g., A865V)²⁷ Amino acid position 866 (e.g., S866A)⁵⁷ Amino acidposition 868 (e.g., V868D)⁷³ Amino acid position 869 (e.g., Q869P)⁷³Amino acid position 875 (e.g., Q875X)⁷³ Amino acid position 877 (e.g.,G877R)⁵⁶ Amino acid position 879 (e.g., I879R)⁸⁸ Amino acid position 893(e.g., A893V)⁵⁷ Amino acid position 901 (e.g., S901R¹⁷, S901I⁷³) Aminoacid position 903 (e.g., V903G)⁵⁷ Δ Amino acid position 919¹² Amino acidposition 923 (e.g., T923P)^(2,7) Amino acid position 926 (e.g.,A926P)^(2,7) Amino acid position 928 (e.g., R928X¹⁵, R928Q⁴⁰) Amino acidposition 930 (e.g., K930X⁵, K930Efs*79^(5,10), K930Efs*49²⁶) Amino acidposition 931 (e.g., Q931P)²⁷ Amino acid position 945 (e.g., S945N)⁵⁷Amino acid position 948 (e.g., R948C)^(5,7,26) Amino acid position 958(e.g., R958Q)²⁸ Amino acid position 969 (e.g., K969K)⁸⁸ Δ Amino acidpositions 969-972⁵ Amino acid position 973 (e.g., T973I)⁵⁷ Amino acidposition 976 (e.g., Q976R⁵⁸, Q976X⁸⁸) Amino acid position 979 (e.g.,N979D)^(5,7) Amino acid position 981 (e.g., Y981Y)²⁸ Amino acid position982 (e.g., G982R)^(2,5,7) Amino acid positions 444 and 982 (e.g.,V444A + G982R)³⁸ Amino acid position 995 (e.g., A995A)²⁸ Amino acidposition 1001 (e.g., R1001R)⁹ Amino acid position 1003 (e.g., G1003R)²⁴Amino acid position 1004 (e.g., G1004D)^(2,7) Amino acid position 1027(e.g., S1027R)²⁶ Amino acid position 1028 (e.g., A1028A^(7,10,88),A1028E⁸⁸) Amino acid position 1029 (e.g., T1029K)⁵ Amino acid position1032 (e.g., G1032R)¹² Amino acid position 1041 (e.g., Y1041X)⁹ Aminoacid position 1044 (e.g., A1044P)⁸⁸ Amino acid position 1050 (e.g.,R1050C)^(2,7,57) Amino acid position 1053 (e.g., Q1053X)⁵⁷ Amino acidposition 1055 (e.g., L1055P)³⁶ Amino acid position 1057 (e.g., R1057X²,R1057Q⁵⁸) Amino acid position 1058 (e.g., Q1058Hfs*38⁹, Q1058fs*38¹⁷,Q1058X⁷³) Amino acid position 1061 (e.g., I1061Vfs*34)⁹ Amino acidposition 1083 (e.g., C1083Y)⁴⁷ Amino acid position 1086 (e.g., T1086T)²⁸Amino acid position 1090 (e.g., R1090X)^(2,5) Amino acid position 1099(e.g., L1099Lfs*38)²⁶ Amino acid position 1100 (e.g., S1100Qfs*38)¹³Amino acid position 1110 (e.g., A1110E)^(5,7) Amino acid position 1112(e.g., V1112F)⁷⁰ Amino acid position 1116 (e.g., G1116R⁷, G1116F^(9,17),G1116E³⁶) Amino acid position 1120 (e.g., S1120N)⁸⁸ Amino acid position1128 (e.g., R1128H^(2,7), R1128C^(5,7,13)) Amino acid position 1131(e.g., D1131V)²⁷ Amino acid position 1144 (e.g., S1144R)⁷ Amino acidposition 1147 (e.g., V1147X)⁵ Amino acid position 1153 (e.g.,R1153C^(2,5,7), R1153H⁵) Amino acid position 1154 (e.g., S1154P)^(5,7)Amino acid position 1162 (e.g., E1162X)³⁹ Δ Amino acid position 1165⁸⁸Amino acid position 1164 (e.g., V1164Gfs*7) Amino acid position 1173(e.g., N1173D)⁵⁷ Amino acid position 1175 (e.g., K1175T)⁵⁸ Amino acidposition 1186 (e.g., E1186K)⁷ Amino acid position 1192 (e.g.,A1192Efs*50)⁹ Amino acid position 1196 (e.g., Q1196X)⁸⁸ Amino acidposition 1197 (e.g., L1197G)⁷ Amino acid position 1198 (e.g., H1198R)²⁷Amino acid position 1204 (e.g., L1204P)⁸⁸ Amino acid position 1208 (e.g.Y1208C)⁷³ Amino acid position 1210 (e.g., T1210P^(5,7), T1210F⁵⁷) Aminoacid position 1211 (e.g., N1211D)⁷ Amino acid position 1212 (e.g.,V1212F)³⁶ Amino acid position 1215 (e.g., Q1215X)⁵ Amino acid position1221 (e.g., R1221K)⁵³ Amino acid position 1223 (e.g., E1223D)⁷ Aminoacid position 1226 (e.g., R1226P)⁷³ Amino acid position 1228 (e.g.,A1228V)⁷ Amino acid position 1231 (e.g., R1231W^(5,7), R1231Q^(5,7))Amino acid position 1232 (e.g., A1232D)¹⁷ Amino acid position 1235(e.g., R1235X)^(5,12) Amino acid position 1242 (e.g., L1242I)^(5,7)Amino acid position 1243 (e.g., D1243G)⁶⁷ Amino acid position 1249(e.g., L1249X)⁷³ Amino acid position 1256 (e.g., T1256fs*1296)⁸³ Aminoacid position 1268 (e.g., R1268Q)^(2,7) Amino acid position 1276 (e.g.,R1276H)³⁰ Amino acid position 1283 (e.g., A1283A²⁸, A1283V⁸⁸) Amino acidposition 1292 (e.g., G1292V)⁷³ Amino acid position 1298 (e.g., G1298R)⁵Amino acid position 1302 (e.g., E1302X)⁵ Amino acid position 1311 (e.g.,Y1311X)⁵⁷ Amino acid position 1316 (e.g., T1316Lfs*64)¹⁵ Amino acidposition 1321 (e.g., S1321N)⁵⁷ Intron 4 ((+3)A > C)¹ IVS4 − 74A > T⁸⁹Splice site mutation 3′ Intron 5 c.3901G > A⁵ Splice site mutation 5;Intron 7 c.6111G > A⁵ Splice site mutation IVS7 + 1G > A¹⁴ IVS7 + 5G >A⁴⁰ IVS8 + 1G > C⁷⁶ Splice site mutation 5′ Intron 9 c.9081delG⁵ Splicesite mutation 5′ Intron 9 c.9081G > T⁵ Splice site mutation 5′ Intron 9c.9081G > A⁵ Splice site mutation IVS9 + 1G > T¹⁴ Splice site mutation3′ Intron 13 c.143513_1435-8del⁵ Splice site mutationIVS13del-13{circumflex over ( )}-8¹⁴ Splice site mutation 3′ Intron 16c.20128T > G⁵ Splice site mutation IVS16 − 8T > G¹⁴ Splice site mutation5′ Intron 18 c.21781G > T⁵ Splice site mutation 5′ Intron 18 c.21781G >A⁵ Splice site mutation 5′ Intron 18 c.21781G > C⁵ Splice site mutation3′ Intron 18 c.21792A > G⁵ Splice site mutation IVS18 + 1G > A¹⁴ Splicesite mutation 5′ Intron 19 c.2343 + 1G > T⁵ Splice site mutation 5′Intron 19 c.2343 + 2T > C⁵ Splice site mutation IVS19 + 2T > C¹⁴ Splicesite mutation IVS19 + 1G > A²² Splice site mutation 3′ Intron 21c.26112A > T⁵ IVS22 + 3A > G⁸⁹ IVS 23 − 8 G − A³⁶ IVS24 + 5G > A⁵¹Splice site mutation 5′ Intron 24 c.32131delG⁵ IVS35 − 6C > G⁸⁹ Putativesplice mutation 1198 − 1G > C¹⁷ Putative splice mutation 1810 − 3C > G¹⁷Putative splice mutation 2178 + 1G > A¹⁷ Putative splice mutation 2344 −1G > T¹⁷ Putative splice mutation c.2611 − 2A > T³⁹ Putative splicemutation 3213 + 1_3213 + 2delinsA¹⁷ c.-24C > A^(44,78) c.76 13 G > T⁹c.77 − 19T > A⁵² c.90_93delGAAA¹⁸ c.124G > A⁶⁹ c.150 + 3 A > C¹⁰ 174C >T⁵⁴ c.245T > C⁸⁷ c.249_250insT¹⁸ 270T > C⁵⁴ 402C > T⁵⁴ 585G > C⁵⁴c.611 + 1G > A⁷⁰ c.611 + 4A > G³⁶ c.612 − 15_-6del10bp⁵⁵ c.625A > C³¹c.627 + 5G > T³¹ c.625A > C/c.627 + 5G > T³¹ 696G > T⁵⁴ c. 784 + 1G >C⁴⁹ 807T > C⁵⁴ c.886C > T³¹ c.890A > G⁵⁹ c.908 + 1G > A⁵⁷ c.908 + 5G >A⁵⁵ c.908delG⁵⁹ c.909 − 15A > G⁶⁶ 957A > G⁵⁴ c.1084 − 2A > G⁵⁷ 1145 1 bpdeletion⁹⁰ 1281C > T^(54,57) c.1309 − 165C > T¹⁹ c.1434 + 174G > A¹⁹c.1434 + 70C > T¹⁹ c.1530C > A⁵⁷ c.1587 − 1589delCTT³¹ c.1621A >C^(33,59) c.1638 + 32T > C⁶⁶ c.1638 + 80C > T⁶⁶ 1671C > T⁵⁴ 1791G > T⁵⁴1939delA¹⁴ c.2075 + 3A > G⁵³ c.2081T > A³¹ c.2093G > A⁶⁵ 2098delA¹⁶c.2138 − 8T > G⁶⁷ 2142A > G⁵⁴ c.2178 + 1G > T^(36,39) c.2179 − 17C > A⁶⁶c.2344 − 157T > G⁶⁶ c.2344 − 17T > C⁶⁶ c.2417G > A⁷⁸ c.2541delG⁸⁷c.2620C > T^(32,33) c.2815 − 8A > G⁵⁵ c.3003A > G³⁷ c.3084A > G^(48,54)c.3213 + 4 A > G^(9,37) c.3213 + 5 G > A⁹ c.3268C > T⁷⁵ 3285A > G⁵⁴c.3382C > T⁷⁵ 3435A > G⁵⁴ c.3491delT⁷² c.3589C > T⁵⁷ c.3765(+1 +5)del5⁴² c.3766 − 34A > G⁶⁶ c.3767 − 3768insC⁶ c.3770delA⁶⁷ c.3826C >T⁷² c.3846C > T⁵⁷ c.3929delG⁶⁷ c.*236A > G⁶⁶ 1145delC⁸ Ex13_Ex17del⁸²

TABLE 4 Selected ABCB11 Mutations Associated with PFIC-2 Amino acidposition 1 (e.g., M1V)⁹ Amino acid position 4 (e.g., S4X)⁶⁴ Amino acidposition 19 (e.g., G19R)⁵⁶ Amino acid position 25 (e.g., S25X)¹⁴ Aminoacid position 26 (e.g., Y26Ifs*7)³⁸ Amino acid position 50 (e.g.,L50S)^(7,57) Amino acid position 52 (e.g., R52W)²⁶ Amino acid position58 (e.g., D58N)⁶² Amino acid position 62 (e.g., M62K)⁹ Amino acidposition 66 (e.g., S66N)¹⁷ Amino acid position 68 (e.g., C68Y)⁴¹ Aminoacid position 93 (e.g., Y93S)¹³ Amino acid position 101 (e.g.,Q101Dfs*8)⁹ Amino acid position 107 (e.g., C107R)³⁶ Amino acid position112 (e.g., I112T)⁹ Amino acid position 114 (e.g., W114R)^(2,9) Aminoacid position 129 (e.g., C129Y)²⁵ Amino acid position 135 (e.g.,E135K¹³, E135L¹⁷) Amino acid position 167 (e.g., A167V⁷, A167T^(9,17))Amino acid position 182 (e.g., I182K)⁹ Amino acid position 183 (e.g.,M183V⁸, M183T⁹) Amino acid position 225 (e.g., T225P)⁵⁷ Amino acidposition 226 (e.g., S226L)⁹ Amino acid position 232 (e.g., L232Cfs*9)⁹Amino acid position 233 (e.g., L233S)⁸⁶ Amino acid position 238 (e.g.,G238V)^(2,7) Amino acid position 242 (e.g., T242I)⁷ Amino acid position245 (e.g., I245Tfs*26)⁵⁷ Amino acid position 256 (e.g., A256G)⁹ Aminoacid position 260 (e.g., G260D)⁵⁷ Amino acid position 284 (e.g., V284L)⁷Amino acid position 297 (e.g., E297G)^(2,7) Amino acid position 303(e.g., R303K⁸, R303M⁶³, R303fsX321⁸³) Amino acid position 304 (e.g.,Y304X)²⁶ Amino acid position 312 (e.g., Q312H)⁷ Amino acid position 313(e.g., R313S)⁷ Amino acid position 314 (e.g., W314X)⁵⁷ Amino acidposition 318 (e.g., K318Rfs*26)²⁹ Amino acid position 327 (e.g., G327E)⁷Amino acid position 330 (e.g., V330X)²⁴ Amino acid position 336 (e.g.,C336S)^(2,7) Amino acid position 337 (e.g., Y337H)²¹ Amino acid position342 (e.g., W342G)⁵⁰ Amino acid position 354 (e.g., R354X)⁹ Amino acidposition 361 (e.g., Q361X)⁵⁷ Amino acid position 366 (e.g., V366D)⁵⁷Amino acid position 386 (e.g., G386X)³⁴ Δ Amino acid positions 383-389⁵⁷Amino acid position 387 (e.g., R387H)⁹ Amino acid position 390 (e.g.,A390P)⁷ Amino acid position 410 (e.g., G410D)⁷ Amino acid position 413(e.g., L413W)⁷ Amino acid position 415 (e.g., R415X)⁴² Amino acidposition 420 (e.g., I420T)⁹ Amino acid position 454 (e.g., V454X)⁴⁹Amino acid position 455 (e.g., G455E)⁹ Amino acid position 461 (e.g.,K461E)^(2,7) Amino acid position 463 (e.g., T463I)⁷ Amino acid position466 (e.g., Q466K)⁷ Amino acid position 470 (e.g., R470Q⁷, R470X⁹) Aminoacid position 472 (e.g., Y472X¹⁴, Y472C²⁷) Amino acid position 475(e.g., C475X)²⁹ Amino acid position 481 (e.g., V481E)⁷ Amino acidposition 482 (e.g., D482G)^(2,7) Amino acid position 484 (e.g.,H484Rfs*5)⁹ Amino acid position 487 (e.g., R487H², R487P⁸⁴) Amino acidposition 490 (e.g., N490D)⁷ Amino acid position 493 (e.g., W493X)⁸ Aminoacid position 498 (e.g., I498T)⁷ Amino acid position 501 (e.g., V501G)⁶⁸Amino acid position 512 (e.g., I512T)⁷ Amino acid position 515 (e.g.,N515T⁷, N515D⁶⁴) Amino acid position 516 (e.g., I516M)¹⁷ Amino acidposition 517 (e.g., R517H)⁷ Amino acid position 520 (e.g., R520X)⁵⁷Amino acid position 523 (e.g., A523G)¹³ Amino acid position 528 (e.g.,I528X)⁹ Amino acid position 540 (e.g., F540L)⁴⁶ Amino acid position 541(e.g., I541L⁷, I541T¹⁷) Amino acid position 548 (e.g., F548Y)⁷ Aminoacid position 549 (e.g., D549V)⁹ Amino acid position 554 (e.g., E554K)²¹Amino acid position 559 (e.g., M559T)⁵⁷ Amino acid position 562 (e.g.,G562D)⁷ Amino acid position 570 (e.g., A570T⁷, A570V²⁶) Amino acidposition 575 (e.g., R575X², R575Q²¹) Amino acid position 588 (e.g.,A588V)⁷ Amino acid position 591 (e.g., N591S)^(9,17) Amino acid position593 (e.g., S593R)^(2,7) Amino acid position 597 (e.g., V597V⁹, V597L¹³)Amino acid positions 591 and 597 (e.g., N591S + V597V)⁹ Amino acidposition 603 (e.g., K603K)⁵⁵ Amino acid position 609 (e.g.,H609Hfs*46)²⁶ Amino acid position 610 (e.g., I610Gfs*45)⁹ Amino acidposition 615 (e.g., H615R)²⁶ Amino acid position 625 (e.g., T625Nfs*5)²⁶Amino acid position 627 (e.g., I627T)⁷ Amino acid position 636 (e.g.,E636G)² Amino acid position 669 (e.g., I669V)²⁶ Amino acid position 698(e.g., R609H)⁹ Amino acid positions 112 and 698 (e.g., I112T + R698H)⁹Amino acid position 699 (e.g., S699P)⁹ Amino acid position 766 (e.g.,G766R)²⁴ Amino acid position 806 (e.g., G806G)⁵⁵ Amino acid position 824(e.g., G824E)⁴² Amino acid position 832 (e.g., R832C^(7,26), R832H⁴¹)Amino acid position 842 (e.g., D842G)² Amino acid position 859 (e.g.,T859R)⁷ Amino acid position 865 (e.g., A865V)⁴⁵ Amino acid position 877(e.g., G877R)⁵⁶ Amino acid position 893 (e.g., A893V)⁵⁷ Amino acidposition 901 (e.g., S901R)¹⁷ Amino acid position 903 (e.g., V903G)⁵⁷ ΔAmino acid position 919¹² Amino acid position 928 (e.g., R928X)^(15,21)Amino acid position 930 (e.g., K930Efs*79¹⁰, K930Efs*49²⁶) Amino acidposition 948 (e.g., R948C)^(7,26) Amino acid position 979 (e.g., N979D)⁷Amino acid position 982 (e.g., G982R)^(2,7) Amino acid positions 444 and982 (e.g., V444A + G982R)³⁸ Amino acid position 1001 (e.g., R1001R)⁹Amino acid position 1003 (e.g., G1003R)²⁴ Amino acid position 1004(e.g., G1004D)^(2,7) Amino acid position 1027 (e.g., S1027R)²⁶ Aminoacid position 1028 (e.g., A1028A)¹⁰ Amino acid position 1032 (e.g.,G1032R)¹² Amino acid position 1041 (e.g., Y1041X)⁹ Amino acid position1050 (e.g., R1050C)⁵⁷ Amino acid position 1053 (e.g., Q1053X)⁵⁷ Aminoacid position 1055 (e.g., L1055P)³⁶ Amino acid position 1057 (e.g.,R1057X)² Amino acid position 1058 (e.g., Q1058Hfs*38⁹, Q1058fs*38¹⁷)Amino acid position 1061 (e.g., I1061Vfs*34)⁹ Amino acid position 1083(e.g., C1083Y)⁴⁷ Amino acid position 1090 (e.g., R1090X)² Amino acidposition 1099 (e.g., L1099Lfs*38)²⁶ Amino acid position 1100 (e.g.,S1100Qfs*38)¹³ Amino acid position 1110 (e.g., A1110E)⁷ Amino acidposition 1116 (e.g., G1116R⁷, G1116F^(9,17), G1116E³⁶) Amino acidposition 1128 (e.g., R1128C)^(7,13) Amino acid position 1131 (e.g.,D1131V)²⁷ Amino acid position 1144 (e.g., S1144R)⁷ Amino acid position1153 (e.g., R1153C^(2,7), R1153H^(7,26)) Amino acid position 1154 (e.g.,S1154P)⁷ Amino acid position 1173 (e.g., N1173D)⁵⁷ Amino acid position1192 (e.g., A1192Efs*50)⁹ Amino acid position 1198 (e.g., H1198R)²⁷Amino acid position 1210 (e.g., T1210P⁷, T1210F⁵⁷) Amino acid position1211 (e.g., N1211D)⁷ Amino acid position 1212 (e.g., V1212F)³⁶ Aminoacid position 1231 (e.g., R1231W⁷, R1223Q⁷) Amino acid position 1232(e.g., A1232D)¹⁷ Amino acid position 1235 (e.g., R1235X)¹² Amino acidposition 1242 (e.g., L1242I)⁷ Amino acid position 1256 (e.g.,T1256fs*1296)⁸³ Amino acid position 1268 (e.g., R1268Q)^(2,7) Amino acidposition 1302 (e.g. E1302X)⁵⁷ Amino acid position 1311 (e.g., Y1311X)⁵⁷Amino acid position 1316 (e.g., T1316Lfs*64)¹⁵ Intron 4 ((+3)A > C)¹Splice site mutation IVS7 + 1G > A¹⁴ IVS8 + 1G > C⁷⁶ Splice sitemutation IVS9 + 1G > T¹⁴ Splice site mutation IVS13del-13{circumflexover ( )}-8¹⁴ Splice site mutation IVS16 − 8T > G¹⁴ Splice site mutationIVS18 + 1G > A¹⁴ Splice site mutation IVS19 + 2T > C¹⁴ IVS 23 − 8 G −A³⁶ IVS24 + 5G > A⁵¹ Putative splice mutation 1198 − 1G > C¹⁷ Putativesplice mutation 1810 − 3C > G¹⁷ Putative splice mutation 2178 + 1G > A¹⁷Putative splice mutation 2344 − 1G > T¹⁷ Putative splice mutation 3213 +1_3213 + 2delinsA¹⁷ c.-24C > A⁷⁸ c.76 13 G > T⁹ c.77 − 19T > A⁵²c.90_93delGAAA¹⁸ c.124G > A⁶⁹ c.150 + 3 A > C¹⁰ c.249_250insT¹⁸ c.611 +1G > A⁸⁴ c.611 + 4A > G³⁶ c.612 − 15_-6del10bp⁵⁵ c.625A > C³¹ c.627 +5G > T³¹ c.625A > C/c.627 + 5G > T³¹ c.886C > T³¹ c.890A > G⁵⁹ c.908 +1G > A⁵⁷ c.908 + 5G > A⁵⁵ c.908delG⁵⁹ 1273 1 bp deletion⁹¹ c.1084 − 2A >G⁵⁷ c.1445A > G⁵⁹ c.1587-1589delCTT³¹ c.1621A > C⁵⁹ 1939delA¹⁴ c.2081T >A³¹ 2098delA¹⁶ c.2343 + 1 G > T⁸⁰ c.2178 + 1G > T³⁶ c.2417G > A⁷⁸c.2620C > T³² c.2815 − 8A > G⁵⁵ c.3003A > G³⁷ c.3213 + 4 A > G^(9,37)c.3213 + 5 G > A⁹ c.3268C > T⁷⁵ c.3382C > T⁷⁵ c.3765(+1 + 5)del5⁴²c.3767-3768insC⁶ 1145delC⁸ Ex13_Ex17del⁸² ^(A) A mutation to ‘X’ denotesan early stop codon

REFERENCES FOR TABLES 3 AND 4

-   ¹ Noe et al., J Hepatol. 2005, vol. 43(3), p. 536-543.-   ² Lam et al., Am J Physiol Cell Physiol. 2007, vol. 293(5), p.    C1709-16.-   ³ Stindt et al., Liver Int. 2013, vol. 33(10), p. 1527-1735.-   ⁴ Gao et al., Shandong Yiyao 2012, vol. 52(10), p. 14-16.-   ⁵ Strautnieks et al., Gastroenterology. 2008, vol. 134(4), p.    1203-1214.-   ⁶ Kagawa et al., Am J Physiol Gastrointest Liver Physiol. 2008, vol.    294(1), p. G58-67.-   ⁷ Byrne et al., Hepatology. 2009, vol. 49(2), p. 553-567.-   ⁸ Chen et al., J Pediatr. 2008, vol. 153(6), p. 825-832.-   ⁹ Davit-Spraul et al., Hepatology 2010, vol. 51(5), p. 1645-1655.-   ¹⁰ Dröge et al., Sci Rep. 2016, vol. 6: 24827.-   ¹¹ Lang et al., Pharmacogenet Genomics. 2007, vol. 17(1), p. 47-60.-   ¹² Ellinger et al., World J Gastroenterol. 2017, vol. 23(29),    p.:5295-5303.-   ¹³ Vitale et al., J Gastroenterol. 2018, vol. 53(8), p. 945-958.-   ¹⁴ Knisely et al., Hepatology. 2006, vol. 44(2), p. 478-86.-   ¹⁵ Ellis et al., Hepatology. 2018, vol. 67(4), p. 1531-1545.-   ¹⁶ Lam et al., J Hepatol. 2006, vol. 44(1), p. 240-242.-   ¹⁷ Varma et al., Hepatology 2015, vol. 62(1), p. 198-206.-   ¹⁸ Treepongkaruna et al., World J Gastroenterol. 2009, vol.    15(34), p. 4339-4342.-   ¹⁹ Zarenezhad et al., Hepatitis Monthly: 2017, vol. 17(2); e43500.-   ²⁰ Hayashi et al., Hepatol Res. 2016, vol. 46(2), p. 192-200.-   ²¹ Guorui et al., Linchuang Erke Zazhi 2013, vol. 31(10), 905-909.-   ²² van Mil et al., Gastroenterology. 2004, vol. 127(2), p. 379-384.-   ²³ Anzivino et al., Dig Liver Dis. 2013, vol. 45(3), p. 226-232.-   ²⁴ Park et al., World J Gastroenterol. 2016, vol. 22(20), p.    4901-4907.-   ²⁵ Imagawa et al., J Hum Genet. 2018, vol. 63(5), p. 569-577.-   ²⁶ Giovannoni et al., PLoS One. 2015, vol. 10(12): e0145021.-   ²⁷ Hu et al., Mol Med Rep. 2014, vol. 10(3), p. 1264-1274.-   ²⁸ Lang et al., Drug Metab Dispos. 2006, vol. 34(9), p. 1582-1599.-   ²⁹ Masahata et al., Transplant Proc. 2016, vol. 48(9), p. 3156-3162.-   ³⁰ Holz et al., Hepatol Commun. 2018, vol. 2(2), p. 152-154.-   ³¹ Li et al., Hepatology International 2017, vol. 11, No. 1, Supp.    Supplement 1, pp. S180. Abstract Number: OP284.-   ³² Francalanci et al., Laboratory Investigation 2011, vol. 91, Supp.    SUPPL. 1, pp. 360A. Abstract Number: 1526.-   ³³ Francalanci et al., Digestive and Liver Disease 2010, vol. 42,    Supp. SUPPL. 1, pp. S16. Abstract Number: T.N.5.-   ³⁴ Shah et al., J Pediatr Genet. 2017, vol. 6(2), p. 126-127.-   ³⁵ Gao et al., Hepatitis Monthly 2017, vol. 17(10),    e55087/1-e55087/6.-   ³⁶ Evason et al., Am J Surg Pathol. 2011, vol. 35(5), p. 687-696.-   ³⁷ Davit-Spraul et al., Mol Genet Metab. 2014, vol. 113(3), p.    225-229.-   ³⁸ Maggiore et al., J Hepatol. 2010, vol. 53(5), p. 981-6.-   ³⁹ McKay et al., Version 2. F1000Res. 2013; 2: 32. DOI:    10.12688/f1000research.2-32.v2-   ⁴⁰ Liu et al., Pediatr Int. 2013, vol. 55(2), p. 138-144.-   ⁴¹ Waisbourd-Zinman et al., Ann Hepatol. 2017, vol. 16(3), p.    465-468.-   ⁴² Griffin, et al., Canadian Journal of Gastroenterology and    Hepatology 2016, vol. 2016. Abstract Number: A200. Meeting Info:    2016 Canadian Digestive Diseases Week, CDDW 2016. Montreal, QC,    United States. 26 Feb. 2016-29 Feb. 2016-   ⁴³ Qiu et al., Hepatology 2017, vol. 65(5), p. 1655-1669.-   ⁴⁴ Imagawa et al., Sci Rep. 2017, 7:41806.-   ⁴⁵ Kang et al., J Pathol Transl Med. 2019 May 16. doi:    10.4132/jptm.2019.05.03. [Epub ahead of print]-   ⁴⁶ Takahashi et al., Eur J Gastroenterol Hepatol. 2007, vol.    19(11), p. 942-6.-   ⁴⁷ Shimizu et al., Am J Transplant. 2011, vol. 11(2), p. 394-398.-   ⁴⁸ Krawczyk et al., Ann Hepatol. 2012, vol. 11(5), p. 710-744.-   ⁴⁹ Sharma et al., BMC Gastroenterol. 2018, vol. 18(1), p. 107.-   ⁵⁰ Sattler et al., Journal of Hepatology 2017, vol. 66, No. 1,    Suppl. S, pp. S177. Meeting Info.: International Liver Congress/52nd    Annual Meeting of the    European-Association-for-the-Study-of-the-Liver. Amsterdam,    NETHERLANDS. Apr. 19-23, 2017. European Assoc Study Liver.-   ⁵¹ Jung et al., J Pediatr Gastroenterol Nutr. 2007, vol. 44(4), p.    453-458.-   ⁵² Sciveres. Digestive and Liver Disease 2010, vol. 42, Supp. SUPPL.    5, pp. S329. Abstract Number: CO18. Meeting Info: 17th National    Congress SIGENP. Pescara, Italy. 7 Oct. 2010-9 Oct. 2010-   ⁵³ Sohn et al., Pediatr Gastroenterol Hepatol Nutr. 2019, vol.    22(2), p. 201-206.-   ⁵⁴ Ho et al., Pharmacogenet Genomics. 2010, vol. 20(1), p. 45-57.-   ⁵⁵ Wang et al., Hepatol Res. 2018, vol. 48(7), p. 574-584.-   ⁵⁶ Shaprio et al., J Hum Genet. 2010, vol. 55(5), p. 308-313.-   ⁵⁷ Bounford. University of Birmingham. Dissertation Abstracts    International, (2016) Vol. 75, No. 1C. Order No.: AA110588329.    ProQuest Dissertations & Theses.-   ⁵⁸ Stolz et al., Aliment Pharmacol Ther. 2019, vol. 49(9), p.    1195-1204.-   ⁵⁹ Jankowska et al., J Pediatr Gastroenterol Nutr. 2014, vol.    58(1), p. 92-95.-   ⁶⁰ Kim. Journal of Pediatric Gastroenterology and Nutrition 2016,    vol. 62, Supp. SUPPL. 1, pp. 620. Abstract Number: H-P-045. Meeting    Info: 49th Annual Meeting of the European Society for Paediatric    Gastroenterology, Hepatology and Nutrition, ESPGHAN 2016. Athens,    Greece. 25 May 2016-28 May 2016.-   ⁶¹ Pauli-Magnus et al., Hepatology 2003, vol. 38, No. 4 Suppl. 1,    pp. 518A. print. Meeting Info.: 54th Annual Meeting of the American    Association for the Study of Liver Diseases. Boston, Mass., USA.    Oct. 24-28, 2003. American Association for the Study of Liver    Diseases.-   ⁶² Li et al., Hepatology International 2017, vol. 11, No. 1, Supp.    Supplement 1, pp. S362. Abstract Number: PP0347. Meeting Info: 26th    Annual Conference of the Asian Pacific Association for the Study of    the Liver, APASL 2017. Shanghai, China. 15 Feb. 2017-19 Feb. 2017.-   ⁶³ Rumbo et al., Transplantation 2018, vol. 102, No. 7, Supp.    Supplement 1, pp. S848. Abstract Number: P.752. Meeting Info: 27th    International Congress of The Transplantation Society, T T S 2018.    Madrid, Spain. 30 Jun. 2018-5 Jul. 2018.-   ⁶⁴ Lee et al., Pediatr Gastroenterol Hepatol Nutr. 2017, vol.    20(2), p. 114-123.-   ⁶⁵ Sherrif et al., Liver international: official journal of the    International Association for the Study of the Liver 2013, vol. 33,    No. 8, pp. 1266-1270.-   ⁶⁶ Blackmore et al., J Clin Exp Hepatol. 2013, vol. 3(2), p.    159-161.-   ⁶⁷ Matte et al., J Pediatr Gastroenterol Nutr. 2010, vol. 51(4), p.    488-493.-   ⁶⁸ Lin et al., Zhongguo Dang Dai Er Ke Za Zhi. 2018, vol. 20(9), p.    758-764.-   ⁶⁹ Harmanci et al., Experimental and Clinical Transplantation 2015,    vol. 13, Supp. SUPPL. 2, pp. 76. Abstract Number: P62. Meeting Info:    1st Congress of the Turkic World Transplantation Society. Astana,    Kazakhstan. 20 May 2015-22 May 2015.-   ⁷⁰ Herbst et al., Mol Cell Probes. 2015, vol. 29(5), p. 291-298.-   ⁷¹ Moghadamrad et al., Hepatology. 2013, vol. 57(6), p. 2539-2541.-   ⁷² Holz et al., Zeitschrift fur Gastroenterologie 2016, vol. 54,    No. 8. Abstract Number: KV275. Meeting Info: Viszeralmedizin    2016, 71. Jahrestagung der Deutschen Gesellschaft fur    Gastroenterologie, Verdauungs- und Stoffwechselkrankheiten mit    Sektion Endoskopie—10. Herbsttagung der Deutschen Gesellschaft fur    Allgemein- und Viszeralchirurgie. Hamburg, Germany. 21 Sep. 2016-24    Sep. 2016.-   ⁷³ Wang et al., PLoS One. 2016; vol. 11(4): e0153114.-   ⁷⁴ Hao et al., International Journal of Clinical and Experimental    Pathology 2017, vol. 10(3), p. 3480-3487.-   ⁷⁵ Arnell et al., J Pediatr Gastroenterol Nutr. 2010, vol. 51(4), p.    494-499.-   ⁷⁶ Sharma et al., Indian Journal of Gastroenterology 2017, vol. 36,    No. 1, Supp. Supplement 1, pp. A99. Abstract Number: M-20. Meeting    Info: 58th Annual Conference of the Indian Society of    Gastroenterology, ISGCON 2017. Bhubaneswar, India. 14 Dec. 2017-17    Dec. 2017.-   ⁷⁷ Beauséjour et al., Can J Gastroenterol. 2011, vol. 25(6), p.    311-314.-   ⁷⁸ Imagawa et al., Journal of Pediatric Gastroenterology and    Nutrition 2016, vol. 63, Supp. Supplement 2, pp. S51. Abstract    Number: 166. Meeting Info: World Congress of Pediatric    Gastroenterology, Hepatology and Nutrition 2016. Montreal, QC,    Canada. 5 Oct. 2016-8 Oct. 2016.-   ⁷⁹ Peng et al., Zhonghua er ke za zhi (Chinese journal of    pediatrics) 2018, vol. 56, No. 6, pp. 440-444.-   ⁸⁰ Tibesar et al., Case Rep Pediatr. 2014, vol. 2014: 185923.-   ⁸¹ Ng et al., Journal of Pediatric Gastroenterology and Nutrition    2018, vol. 66, Supp. Supplement 2, pp. 860. Abstract Number:    H-P-127. Meeting Info: 51st Annual Meeting European Society for    Paediatric Gastroenterology, Hepatology and Nutrition, ESPGHAN 2018.    Geneva, Switzerland. 9 May 2018-12 May 2018.-   ⁸² Wong et al., Clin Chem. 2008, vol. 54(7), p. 1141-1148.-   ⁸³ Pauli-Magnus et al., J Hepatol. 2005, vol. 43(2), p. 342-357.-   ⁸⁴ Jericho et al., Journal of Pediatric Gastroenterology and    Nutrition. 60, vol. 3, p. 368-374.-   ⁸⁵ Scheimann et al., Gastroenterology 2007, vol. 132, No. 4, Suppl.    2, pp. A452. Meeting Info.: Digestive Disease Week Meeting/108th    Annual Meeting of the American-Gastroenterological-Association.    Washington, D.C., USA. May 19-24, 2007. Amer Gastroenterol Assoc;    Amer Assoc Study Liver Dis; Amer Soc Gastrointestinal Endoscopy; Soc    Surg Alimentary Tract.-   ⁸⁶ Jaquotot-Haerranz et al., Rev Esp Enferm Dig. 2013, vol.    105(1), p. 52-54.-   ⁸⁷ Khosla et al., American Journal of Gastroenterology 2015, vol.    110, No. Suppl. 1, pp. S397. Meeting Info.: 80th Annual Scientific    Meeting of the American-College-of-Gastroenterology. Honolulu, Hi.,    USA. Oct. 16-21, 2015.-   ⁸⁸ Dróge et al., J Hepatol. 2017, vol. 67(6), p. 1253-1264.-   ⁸⁹ Liu et al., Liver International 2010, vol. 30(6), p. 809-815.-   ⁹⁰ Chen et al., Journal of Pediatrics 2002, vol. 140(1), p. 119-124.-   ⁹¹ U.S. Pat. No. 9,295,677

In some embodiments, the mutation in ABCB11 is selected from A167T,G238V, V284L, E297G, R470Q, R470X, D482G, R487H, A570T, N591S, A865V,G982R, R1153C, and R1268Q.

Provided are methods of treating PFIC (e.g., PFIC-1 and PFIC-2) in asubject that includes performing an assay on a sample obtained from thesubject to determine whether the subject has a mutation associated withPFIC (e.g., a ATP8B1, ABCB11, ABCB4, TJP2, NR1H4 or Myo5b mutation), andadministering (e.g., specifically or selectively administering) atherapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof, to the subject determined tohave a mutation associated with PFIC. In some embodiments, the mutationis an ATP8B1 or ABCB11 mutation. For example, a mutation as provided inany one of Tables 1-4. In some embodiments, the mutation in ATP8B1 isselected from L127P, G308V, T456M, D554N, F529del, I661T, E665X, R930X,R952X, R1014X, and G1040R. In some embodiments, the mutation in ABCB11is selected from A167T, G238V, V284L, E297G, R470Q, R470X, D482G, R487H,A570T, N591S, A865V, G982R, R1153C, and R1268Q.

Also provided are methods for treating PFIC (e.g., PFIC-1 and PFIC-2) ina subject in need thereof, the method comprising: (a) detecting amutation associated with PFIC (e.g., a ATP8B1, ABCB11, ABCB4, TJP2,NR1H4 or Myo5b mutation) in the subject; and (b) administering to thesubject a therapeutically effective amount of crystal modification I ofodevixibat. In some embodiments, methods for treating PFIC can includeadministering a therapeutically effective amount of a compound offormula (I), or a pharmaceutically acceptable salt thereof, to a subjecthaving a mutation associated with PFIC (e.g., an ATP8B1, ABCB11, ABCB4,TJP2, NR1H4 or Myo5b mutation). In some embodiments, the mutation is anATP8B1 or ABCB11 mutation. For example, a mutation as provided in anyone of Tables 1-4. In some embodiments, the mutation in ATP8B1 isselected from L127P, G308V, T456M, D554N, F529del, I661T, E665X, R930X,R952X, R1014X, and G1040R. In some embodiments, the mutation in ABCB11is selected from A167T, G238V, V284L, E297G, R470Q, R470X, D482G, R487H,A570T, N591S, A865V, G982R, R1153C, and R1268Q.

In some embodiments, the subject is determined to have a mutationassociated with PFIC in a subject or a biopsy sample from the subjectthrough the use of any art recognized tests, including next generationsequencing (NGS). In some embodiments, the subject is determined to havea mutation associated with PFIC using a regulatory agency-approved,e.g., FDA-approved test or assay for identifying a mutation associatedwith PFIC in a subject or a biopsy sample from the subject or byperforming any of the non-limiting examples of assays described herein.Additional methods of diagnosing PFIC are described in Gunaydin, M. etal., Hepat Med. 2018, vol. 10, p. 95-104, incorporated by reference inits entirety herein.

In some embodiments, the treatment of PFIC (e.g., PFIC-1 or PFIC-2)decreases the level of serum bile acids in the subject. In someembodiments, the level of serum bile acids is determined by, forexample, an ELISA enzymatic assay or the assays for the measurement oftotal bile acids as described in Danese et al., PLoS One. 2017, vol.12(6): e0179200, which is incorporated by reference herein in itsentirety. In some embodiments, the level of serum bile acids candecrease by, for example, 10% to 40%, 20% to 50%, 30% to 60%, 40% to70%, 50% to 80%, or by more than 90% of the level of serum bile acidsprior to administration of crystal modification I of odevixibat. In someembodiments, the treatment of PFIC includes treatment of pruritus.

Thus, in one embodiment, the invention relates to crystal modification 1of odevixibat described herein for use in the treatment or prevention ofa disease or disorder as listed above.

In another embodiment, the invention relates to the use of crystalmodification 1 of odevixibat described herein in the manufacture of amedicament for the treatment or prevention of a disease or disorder aslisted above.

In yet another embodiment, the invention relates to a method oftreatment or prevention of a disease or disorder as listed above in awarm-blooded animal, comprising administering a therapeuticallyeffective amount of crystal modification 1 of odevixibat describedherein to a warm-blooded animal in need of such treatment and/orprophylaxis.

Another aspect of the invention relates to a pharmaceutical compositioncomprising a therapeutically effective amount of crystal modification 1of odevixibat described herein, in association with a pharmaceuticallyacceptable diluent or carrier.

The pharmaceutical composition may further comprise at least one otheractive substance, such as an active substance selected from an IBATinhibitor; an enteroendocrine peptide or enhancer thereof; a dipeptidylpeptidase-IV inhibitor; a biguanidine; an incretin mimetic; athiazolidinone; a PPAR agonist; a HMG Co-A reductase inhibitor; a bileacid binder; a TGR5 receptor modulator; a member of the prostone classof compounds; a guanylate cyclase C agonist; a 5-HT4 serotonin agonist;or a pharmaceutically acceptable salt of any one these activesubstances. Examples of such combinations are also described inWO2012/064268.

Crystal modification 1 of odevixibat can be administered to awarm-blooded animal at a unit dose within the range of about 0.01 to 1.0mg/kg, such as about 0.01 to 0.5 mg/kg, or such as about 0.01 to 0.2mg/kg, and this can provide a therapeutically effective dose. A unitdose form, such as a tablet or capsule, can contain about 0.1 to 20 mgof active ingredient, such as about 0.1 to 10 mg, or such as about 0.2to 5 mg, or such as about 0.2 to 1.0 mg. The daily dose can beadministered as a single dose or divided into one, two, three or moreunit doses. An orally administered daily dose of odevixibat ispreferably within about 0.1 to 50 mg, more preferably within about 0.1to 20 mg, such as within about 0.2 to 10 mg, or such as within about 0.2to 5.0 mg.

Pharmaceutical formulations of odevixibat may comprise a therapeuticallyeffective amount of crystal modification 1 of odevixibat, and one ormore pharmaceutically acceptable excipients. The excipients may e.g.include fillers, binders, disintegrants, glidants and lubricants. Ingeneral, pharmaceutical compositions may be prepared in a conventionalmanner using conventional excipients.

In some embodiments, the pharmaceutical formulation is amultiparticulate formulation containing low doses of crystalmodification 1 of odevixibat. Such a formulation enables weight-baseddosing and may be particularly suitable for administering to paediatricpatients. In some embodiments, the pharmaceutical formulation is apaediatric formulation.

In some embodiment, the particles are small enough that they can besprinkled onto food and easily swallowed. In some embodiments, theparticles can be swallowed without causing a perception of grittiness.In some embodiments, the particles do not give the patient an urge tochew the particles.

In some embodiments, each particle comprises a core and a coating layersurrounding the core. The core of each particle may be a pellet, agranule, a minitablet, a bead, a microparticle or a microsphere. Theactive pharmaceutical ingredient may be in the core or in the coatinglayer. In some embodiments, the coating layer of each particle comprisesthe active pharmaceutical ingredient, while the core of each particledoes not comprise the active pharmaceutical ingredient.

The cores may be orally dispersible and comprise soluble ingredientssuch as a sugar (e.g., sucrose) or a soluble polymer (e.g. hydroxypropylmethylcellulose) or may be non-orally dispersible and comprisenon-soluble ingredients such as a non-soluble polymer (e.g.,microcrystalline cellulose). In some embodiments, the cores aremicrocrystalline cellulose spheres.

The coating layer can further comprise a film-forming polymer, such as acellulose-based polymer, a polysaccharide-based polymer, anN-vinylpyrrolidone-based polymer, an acrylate, an acrylamide, orcopolymers thereof. Examples of suitable film-forming polymers includepolyvinyl alcohol (PVA), polyvinyl acetate phthalate (PVAP),polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), methacrylic acidcopolymers, starch, hydroxypropyl starch, chitosan, shellac, methylcellulose, hydroxypropyl cellulose (HPC), low-substituted hydroxypropylcellulose, hydroxypropyl methylcellulose (HPMC; or hypromellose),hydroxypropyl methylcellulose acetate succinate (HPMCAS), hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP),cellulose acetate trimellitate (CAT), as well as combinations thereof,such as a mixture of methyl cellulose and hydroxypropyl methylcellulose(metolose). In some embodiments, the coating layer comprises afilm-forming polymer selected from the group consisting of hydroxypropylmethylcellulose, polyvinyl alcohol (PVA), polyethylene glycol (PEG),starch, hydroxypropyl starch and hydroxypropyl cellulose (HPC).

The coating layer may optionally comprise one or more additionalingredients, such as a plasticizer (e.g. polyethylene glycol, triacetinor triethyl citrate), an anti-tack agent (e.g. talc or magnesiumstearate) or a colouring agent (e.g. titanium dioxide, iron oxides,riboflavin or turmeric).

The dosage required for the therapeutic or prophylactic treatment willdepend on the route of administration, the severity of the disease, theage and weight of the patient and other factors normally considered bythe attending physician when determining the individual regimen anddosage levels appropriate for a particular patient.

Definitions

The term “crystal modification” refers to a crystalline solid phase ofan organic compound. A crystal modification can be either a solvate oran ansolvate.

The term “solvate” refers to a crystalline solid phase of an organiccompound, which has solvent (i.e., solvent molecules) incorporated intoits crystal structure. A “hydrate” is a solvate wherein the solvent iswater.

The term “sesquihydrate” refers to a hydrate containing about 1.5 molesof water associated with the crystal per mole of organic compound (i.e.,a 1.5 hydrate). As used herein, a sesquihydrate includes from about 1.2to about 1.8, more preferably from about 1.3 to about 1.7, morepreferably from about 1.4 to about 1.6 and even more preferably fromabout 1.45 to about 1.55 moles of water associated with each mole ofodevixibat in a crystal. The amount of water calculated herein excludeswater adsorbed to the surface of the crystal.

The term “mixed solvate” refers to a crystalline solid phase of anorganic compound, which has two or more different solvent moleculesincorporated into its crystal structure. One of the at least two solventmolecules may be water.

The term “isostructural solvate” refers to a crystalline solid phase ofan organic compound, wherein the crystalline solid phase can accommodatedifferent solvents without distortion of the crystalline structure.

The term “slurry” refers to a saturated solution to which an excess ofsolid is added, thereby forming a mixture of solid and saturatedsolution.

As used herein, the term “void volumes” refers to channels, layers orother more or less isolated voids in the crystal structure.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

As used herein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions and/or dosage forms that are suitablefor human pharmaceutical use and that are generally safe, non-toxic andneither biologically nor otherwise undesirable.

As used herein, the term “about” refers to a value or parameter hereinthat includes (and describes) embodiments that are directed to thatvalue or parameter per se. For example, description referring to “about20” includes description of “20.” Numeric ranges are inclusive of thenumbers defining the range. Generally speaking, the term “about” refersto the indicated value of the variable and to all values of the variablethat are within the experimental error of the indicated value (e.g.,within the 95% confidence interval for the mean) or within 10 percent ofthe indicated value, whichever is greater.

The crystallinity of a crystalline sample of odevixibat may be measurede.g. by X-Ray Powder Diffraction (XRPD) methods or by DifferentialScanning Calorimetry (DSC) methods, such as the method disclosed in theexperimental section. When reference is made herein to a crystallinecompound, preferably the crystallinity as measured by DSC methods isgreater than about 70%, such as greater than about 80%, particularlygreater than about 90%, more particularly greater than about 95%. Insome embodiments, the degree of crystallinity as measured by DSC methodsis greater than about 98%. In some embodiments, the degree ofcrystallinity as measured by DSC methods is greater than about 99%. The% crystallinity refers to the percentage by weight of the total samplemass which is crystalline.

Preferably a crystal modification according to the invention issubstantially free from other crystal modifications of the compound.Preferably, the described crystal modifications of odevixibat includeless than, for example, about 20%, about 15%, about 10%, about 5%, about3%, or particularly, less than about 1% by weight of other crystalmodifications of odevixibat. Thus, preferably, the solid phase purity ofthe described crystal modifications of odevixibat is greater than about80%, greater than about 85%, greater than about 90%, greater than about95%, greater than about 97%, or particularly greater than about 99%.

The invention will now be described by the following examples which donot limit the invention in any respect. All cited documents andreferences mentioned herein are incorporated by reference in theirentireties.

Abbreviations

-   DMF dimethylformamide-   DMSO dimethyl sulfoxide-   EtOH ethanol-   MeOH methanol-   RH relative humidity-   2-PrOH 2-propanol

EXPERIMENTAL METHODS

X-Ray Powder Diffraction (XRPD) Analysis

Analyses were performed at 22° C. on a PANalytical X'Pert Prodiffractometer equipped with a Cu long fine focus X-ray tube and aPIXcel detector. Automatic divergence and anti-scatter slits were usedtogether with 0.02 rad Soller slits and a Ni-filter. Dry samples weresmeared onto cut Silicon Zero Background Holders (ZBH) and analysedbetween 2-40° in 2-theta with an analysis time of 17 minutes. All slurrysamples were dripped on tempered porous Alumina filter substrates andanalysed twice as they dried, first with a one minute 16-second scan(2-30° in 2-theta) and then a 7-minute scan (2-30° in 2-theta). A final17-minute scan was performed when the sample had dried for severalhours.

The samples were spun during analysis in order to increase therandomness of the samples. The following experimental settings wereused:

Tube tension and current: 40 kV, 50 mA

Wavelength alpha1 (CuKα1): 1.5406 Å

Wavelength alpha2 (CuKα2): 1.5444 Å

Wavelength alpha1 and alpha2 mean (CuKα): 1.5418 Å

It is known in the art that an X-ray powder diffraction pattern may beobtained having one or more measurement errors depending on measurementconditions (such as equipment, sample preparation or machine used). Inparticular, it is generally known that intensities in an XRPD patternmay fluctuate depending on measurement conditions and samplepreparation. For example, persons skilled in the art of XRPD willrealise that the relative intensities of peaks may vary according to theorientation of the sample under the test and on the type and setting ofthe instrument used. The skilled person will also realise that theposition of reflections can be affected by the precise height at whichthe sample sits in the diffractometer and the zero calibration of thediffractometer. The surface planarity of the sample may also have asmall effect. Hence a person skilled in the art will appreciate that thediffraction pattern presented herein is not to be construed as absoluteand any crystalline form that provides a powder diffraction patternsubstantially identical to those disclosed herein fall within the scopeof the present disclosure (for further information, see R. Jenkins andR. L. Snyder, “Introduction to X-ray powder diffractometry”, John Wiley& Sons, 1996).

Thermogravimetric Analysis (TGA)

The analyses were performed on a Mettler TGA/SDTA 851e, equipped with aJulabo FP40 cooler. 1-10 mg of sample was weighed into 100 μL Al-cupsand flushed with dry nitrogen gas during the analysis. Two differentmethods were used: in the “standard scan” the sample was scanned from 25to 200° C. with a scan rate of 10° C./minute, and in the “careful scan”the sample was kept at 25° C. for 30 minutes and was then scanned from25 to 100° C. with a scan rate of 10° C./minute.

Dynamic Vapor Sorption (DVS)

DVS measurements were performed with an SPS11-100n “SorptionsPrufsystem” from ProUmid (formerly “Projekt Messtechnik”),August-Nagel-Str. 23, 89079 Ulm (Germany). About 20 mg of sample wasused. Humidity change rates of 5% per hour were used. The sample wasplaced on an aluminum or platinum holder on top of a microbalance andallowed to equilibrate at 0% RH before starting the pre-defined humidityprogram:

-   -   (1) 5h at 0% RH    -   (2) 0→95% RH (5%/h); 5 h at 95% RH    -   (3) 95→0% RH (5%/h); 5 h at 0% RH    -   (4) 0→95% RH (5%/h); 5 h at 95% RH    -   (5) 95→0% RH (5%/h); 5 h at 0% RH

High-Performance Liquid Chromatography (HPLC)

Analyses were performed on an Agilent, Series 1100, equipped with anAgilent 1260 Infinity degasser. Column: Waters XSelcet CHS C18 (150×3mm, 3.5 am); Mobile phase A: 0.1% formic acid in water, mobile phase B:0.1% formic acid in acetonitrile; Gradient 45% to 90% B; flow rate 0.425mL/min; Acquisition time 35 minutes; Run time 42 minutes; Wave length:283 nm; Column temperature 20° C. The Chromeleon Version 6.8 softwarewas used.

Differential Scanning Calorimetry (DSC)

Experiments were performed using a TA Instruments Q2000 DifferentialScanning Calorimeter. The DCS crucible used was a TZero aluminum panwith pinhole (diameter >0.2 mm) in the lid. A dry nitrogen purge at aconstant flow rate of 50 mL/min was maintained in the DSC cellthroughout the measurement.

EXAMPLES Example 1

Preparation of Crystal Modification 1

Absolute alcohol (100.42 kg) and crude odevixibat (18.16 kg) werecharged to a 250-L GLR with stirring under nitrogen atmosphere. Purifiedwater (12.71 kg) was added and the reaction mass was stirred undernitrogen atmosphere at 25±5° C. for 15 minutes. Stirring was continuedat 25±5° C. for 3 to 60 minutes, until a clear solution had formed. Thesolution was filtered through a 5.0μ SS cartridge filter, followed by a0.2μ PP cartridge filter and then transferred to a clean reactor.Purified water (63.56 kg) was added slowly over a period of 2 to 3 hoursat 25±5° C., and the solution was seeded with crystal modification 1 ofodevixibat. The solution was stirred at 25±5° C. for 12 hours. Duringthis time, the solution turned turbid. The precipitated solids werefiltered through centrifuge and the material was spin dried for 30minutes. The material was thereafter vacuum dried in a Nutsche filterfor 12 hours. The material was then dried in a vacuum tray drier at25±5° C. under vacuum (550 mm Hg) for 10 hours and then at 30±5° C.under vacuum (550 mm Hg) for 16 hours. The material was isolated as anoff-white crystalline solid. The isolated crystalline material wasmilled and stored in LDPE bags.

An overhydrated sample was analyzed with XRPD and the diffractogram isshown in FIG. 2. Another sample was dried at 50° C. in vacuum andthereafter analysed with XRPD. The diffractogram of the dried sample isshown in FIG. 1.

The diffractograms for the drying of the sample are shown in FIGS. 3 and4 for 20 ranges 5-13° and 18-25°, respectively (overhydrated sample atthe bottom and dry sample at the top).

Example 2

Preparation of Crystal Modification 2A from Ethanol and Water

105.9 mg of odevixibat were weighed into a 1 mL Chromacol vessel. Amagnetic stir bar and 1.0 mL of an ethanol:water 70:30% v/v mixture wereadded and the vessel was closed with a crimped cap. The resulting slurrywas then left stirred at 25° C. for 1 week.

The wet sample was analyzed with XRPD and the diffractogram is shown inFIG. 6. Upon drying of the sample, it transformed into crystalmodification 1.

Example 3

Preparation of Crystal Modification 2A from Acetone and Water

27.0 mg of odevixibat were weighed into a 1 mL Chromacol vessel. Amagnetic stir bar and 0.5 mL of a acetone:water 50:50% v/v mixture wereadded and the vessel was closed with a crimped cap. The resulting slurrywas then left stirred at 3° C. for 2 weeks.

The wet sample was analyzed with XRPD and the diffractogram is shown inFIG. 7. Upon drying of the sample, it transformed into crystalmodification 1.

Example 4

Preparation of Crystal Modification 2A from 2-Propanol and Water

27.4 mg of odevixibat were weighed into a 1 mL Chromacol vessel. Amagnetic stir bar and 0.5 mL of a 2-propanol:water 50:50% v/v mixturewere added and the vessel was closed with a crimped cap. The resultingslurry was then left stirred at 3° C. for 2 weeks.

The wet sample was analyzed with XRPD and the diffractogram is shown inFIG. 8. Upon drying of the sample, it transformed into crystalmodification 1.

Example 5

Preparation of Crystal Modification 2A from 1,4-Dioxane and Water

31.6 mg of odevixibat were weighed into a 1 mL Chromacol vessel. Amagnetic stir bar and 0.5 mL of a 1,4-dioxane:water 50:50% v/v mixturewere added and the vessel was closed with a crimped cap. The resultingslurry was then left stirred at 3° C. for 2 weeks.

The wet sample was analyzed with XRPD and the diffractogram is shown inFIG. 9. Upon drying of the sample, it transformed into crystalmodification 1.

Example 6

Preparation of Crystal Modification 2B from Methanol

103.9 mg of odevixibat were weighed into a 1 mL Chromacol vessel. Amagnetic stir bar and 0.9 mL of methanol was added and the vessel wasclosed with a crimped cap. The resulting slurry was then left stirred at22° C. for 1 week.

The wet sample was analyzed with XRPD and the diffractogram is shown inFIG. 9. Upon drying of the sample, it transformed into crystalmodification 1.

Example 7

Preparation of Crystal Modification 2B from Acetonitrile and Water

20.2 mg of odevixibat were dissolved in 1.5 mL acetonitrile. To thestirred solution, 2.5 mL water was added as an antisolvent. Within 20-30minutes a slurry had precipitated.

The wet sample was analyzed with XRPD and the diffractogram is shown inFIG. 10. Upon drying of the sample, it transformed into crystalmodification 1.

Example 8

Preparation of Crystal Modification 2C from DMSO and Water

29.8 mg of odevixibat were weighed into a 1 mL Chromacol vessel. Amagnetic stir bar and 0.5 mL of a DMSO:water 50:50% v/v mixture wereadded and the vessel was closed with a crimped cap. The resulting slurrywas then left stirred at 3° C. for 2 weeks.

The wet sample was analyzed with XRPD and the diffractogram is shown inFIG. 12. Upon drying of the sample, it transformed into crystalmodification 1.

Example 9

Analysis of the Water and Solvent Content of Crystal Modifications 1 and2

Karl-Fischer analysis of crystals of modification 1 showed a watercontent of 3.4% w/w. Thermal gravimetric analysis (TGA) of the samematerial showed a total mass loss of 3.5% (see FIG. 13). These similarfindings indicate that crystal modification 1 contains 1.5 moles ofwater per mole of odevixibat, corresponding to a 1.5 hydrate.

The water and solvent content in crystal modification 2 were analysed byusing samples prepared from a slurry of odevixibat in ethanol:water(60:40% v/v) that had been allowed to equilibrate during 3 days. Form 2had formed according to XRPD. Slurry samples were taken from the slurryto Porous Plates and then stored in a desiccator with ethanol:water(60:40% v/v) and equilibrated at least overnight. Plates were taken outand dried in air for a certain time (5-30 minutes), and then analysedwith a fast scan on XRPD (1 min 16 s) to verify the crystal form. Somesamples contained crystal modification 2 and were still very wet,whereas crystal modification 1 already started to appear in the driersamples. Karl-Fischer analysis of the dried samples of crystalmodification 2 indicated a water content of slightly more than 4% w/w.Thermal gravimetric analysis of the very wet samples of crystalmodification 2 showed that these samples initially lost a lot of mass. Achange in drying rate was thereafter observed, which probably indicatesthe start of the transformation from modification 2 to modification 1.After performing several experiments, a mass loss of approximately 12%w/w could be determined for the transformation of modification 2 tomodification 1. Since dry modification 1 is a sesquihydrate (see FIG.13), the total mass loss of approximately 12% (w/w) for thetransformation of crystal modification 2 to crystal modification 1 wouldcorrespond to a loss of two moles of ethanol and 0.5 moles of water.

In another experiment, a sample of crystal modification 1 was kept in adessicator and exposed to the vapour phase of a 60:40 (% v/v) mixture ofethanol and water for 4 days at room temperature. Thermal gravimetricanalysis of the sample showed a mass loss of about 18.7% (see FIG. 14).The mass loss begins readily at the beginning of the experiment. Furtherexamination of the sample by 1H-NMR suggested that the ethanol contentcorresponded to about 2.7 equivalents and the water content to about 1.9equivalents.

Example 10

Dynamic Vapor Sorption Analysis of Crystal Modification 1

The water uptake of crystal modification 1 was measured using dynamicvapour sorption (DVS). The measurements demonstrate that the watercontent is reversibly dependent on the environmental humidity withmaximum uptakes of about 5.0% (w/w) at 95% RH, as shown in FIG. 15.

After drying the sample at 0% RH and increasing the relative humidity,most of the water was taken back up to about 25% RH. This corresponds toa water content of about 3.5% (w/w). An additional 1.5% (w/w) of waterwas then taken up when the humidity was increased up to 95% RH. Thesorption/desorption process shows minimal hysteresis. XRPD analysis hasshown that the hydrate structure is almost completely restored at 20% RHand is completely restored at 30% RH. Crystal modification 1 thereforeseems to require about 3.5% (w/w) of water, which corresponds to asesquihydrate. The further water uptake at higher relative humiditiesdoes not change the structure any further. Crystal modification 1 istherefore likely a slightly hygroscopic sesquihydrate that can take upadditional 1.5% (w/w) of water at elevated relative humidity in therange of 30-95% RH.

Example 11

Stability Testing

Samples of amorphous odevixibat (purity ^(˜)91%) and of crystalmodification 1 of odevixibat (purity >99%; crystallinity 100%) werestored in a closed container under air at 80° C. The amount ofodevixibat in the samples was determined by HPLC at the beginning of theexperiment, and was again determined after 1, 2 and 4 weeks. The resultsare shown in the table below. After 4 weeks of storage, the amorphoussample showed 0.3% decomposition, whereas the purity of the crystallinesample had not changed.

Odevixibat content (%) Time (weeks) Amorphous odevixibat crystalmodification 1 0 91.1 99.13 1 90.9 99.15 2 91.04 99.18 4 90.8 99.24

Example 12

Determination of Crystalline Fraction by Differential ScanningCalorimetry

This method quantifies the crystalline fraction of odevixibat drugsubstance in partially crystalline samples. The quantification is basedon the assumption that partially crystalline samples are binary mixturesof the crystalline hydrate and the amorphous phase of odevixibat. Thecrystalline fraction is quantified based on the melting enthalpy of ananhydrous form. This anhydrous form is the dehydrated hydrate whichspontaneously and reproducibly forms by drying the hydrate at elevatedtemperature.

5-6 mg of a sample of a crystalline or partially crystalline sample ofodevixibat was accurately weighed into a DSC crucible which was thenclosed with a perforated lid using a suitable press. The total weight ofthe DSC crucible (pan+lid+sample) was noted and the total weight of thecrucible was again determined after the DSC test. The weight loss duringthe DSC test must not be more than 5%.

The DSC test consists of three cycles:

-   -   Cycle 1: an increase in temperature from 20° C. to 120° C. at a        scanning rate of 5° C./min;    -   Cycle 2: a decrease in temperature from 120° C. to 80° C. at a        scanning rate of 10° C./min; and    -   Cycle 3: an increase in temperature from 80° C. to 200° C. at a        scanning rate of 10° C./min.

The first scan cycle dries the sample and thereby converts the hydrateform into a dehydrated hydrate (an anhydrous form). In the second scancycle, the sample is cooled down to obtain a stable baseline in thesubsequent heat-up for signal integration. The melting enthalpy isdetermined in the third scan cycle, where the sample is heated throughthe melting of the anhydrous form. The endothermic event due to meltingappears in the temperature range of 140-165° C. The peak must beintegrated over a sigmoidal tangent baseline using the Sig Tangentintegration function of the TA Universal Analysis software. Theintegration should start at a temperature between 130° C. and 140° C.,and end at a temperature between 165° C. and 175° C., depending on theactual baseline. The glass transition of the amorphous part may appearin the temperature range of 120-130° C., depending on the actualamorphous fraction (see FIG. 16 for an example). If an irregularbaseline does not allow the integration, it should be assessed whetherthe drying of the sample was incomplete.

The evaluation of the melting enthalpy is done by using the dry weightof the sample, which is obtained by subtracting the total weight of theDSC crucible (pan+lid+sample) after the DSC test from the total weightof the crucible before the test. The percent weight loss during the DSCscan, which is the difference between the initial weight and the dryweight divided by the initial weight, must not be more than 5%;otherwise the crystalline content of the sample cannot be calculated.The crystalline fraction expressed in weight percent is to be calculatedfrom the melting enthalpy (ΔH_(sample)) based on the following formula.The value shall be given on an integer number.

${\%\mspace{14mu}{crystalline}\mspace{14mu}{content}} = \frac{{\Delta\; H_{sample}} + 1.1626}{0.2815}$

Example 13 Effect of Drying on the Crystallinity of Crystal Modification1

In these experiments, crystal modification 2 was obtained after slurringof crystal modification 1 in a 6:4 mixture of ethanol/water; theobtained wet material was thereafter stored in a desiccator underethanol/water (6:4) vapor for two months.

Samples of crystal modification 2 were then dried using different dryingtechniques, in order to see the impact of drying on the crystallinity ofcrystal modification 1. The dried samples were analyzed using XRPD(samples were prepared in an ambient air atmosphere) and the results areshown in the table below. The results suggest that crystal modification1 is obtained by rehydration of the dehydrated form, which is obtainedby drying of crystal modification 2 under vacuum or under nitrogen flow.When crystal modification 2 is stored at ambient conditions, theethanol-water exchange seems to be very low.

Drying conditions Results Vacuum (<5 mbar), room temperature. Crystalmodification 1 Nitrogen flow, room temperature Crystal modification 1Ambient conditions Poorly crystalline crystal modification 1

Example 14

Effect of Solvent on Crystallinity of Crystal Modification 2

Crystal modification 1 was suspended in a 30:70 (% v/v) mixture ofethanol and water (sample A) or in a 70:30 (% v/v) mixture of ethanoland water (sample B) at room temperature. After stirring overnight,filtration was conducted and the recovered wet samples were submittedfor XRPD (transmission). The XRPD patterns for both samples essentiallycorresponded to crystal modification 2, but some slight peak shifts wereobserved between the two samples, possibly due to the difference inethanol content of the two samples.

Both samples were then subjected to air drying at room temperature andretested by XRPD. In both cases, crystal modification 1 was obtained,but based on the peak resolution in the XRPD patterns the sampleobtained from the 70:30 (% v/v) mixture of ethanol and water appearedconsiderably more crystalline.

DSC measurements were conducted on the air-dried samples. It was foundthat sample A, obtained from the mixture containing 30% ethanol, wasless crystalline than sample B, obtained from the mixture containing 70%ethanol. An enthalpy of fusion of 25.7 J/g was found for sample A whichcorresponds to 95% of crystallinity. For sample B, an enthalpy of 28.9J/g was found, which corresponds to more than 100% crystallinity.

The invention claimed is:
 1. A crystalline hydrate of odevixibat.
 2. Thehydrate according to claim 1, which is a channel hydrate.
 3. The hydrateaccording to claim 1, which comprises from about 0 to about 2 moles ofwater associated with the crystal per mole of odevixibat.
 4. The hydrateaccording to claim 1, which is a sesquihydrate.
 5. The hydrate accordingto claim 1, having an XRPD pattern, obtained with CuKα1-radiation, withpeaks at °2θ positions 5.6±0.2, 6.7±0.2 and/or 12.1±0.2.
 6. The hydrateaccording to claim 5, having an XRPD pattern, obtained withCuKα1-radiation, with specific peaks at °2θ positions 5.6±0.2, 6.7±0.2and 12.1±0.2 and one or more of the characteristic peaks: 4.1±0.2,4.6±0.2, 9.3±0.2, 9.4±0.2 and 10.7±0.2.
 7. The hydrate according toclaim 5, having a crystallinity of greater than about 99%.
 8. A mixedsolvate of odevixibat, containing about two moles of water per mole ofodevixibat.
 9. The mixed solvate according to claim 8, wherein theorganic solvent is methanol, ethanol, 2-propanol, acetone, acetonitrile,1,4-dioxane, DMF or DMSO.
 10. The mixed solvate according to claim 8,wherein the organic solvent is ethanol.
 11. The mixed solvate accordingto claim 8, having an XRPD pattern, obtained with CuKα1-radiation, withpeaks at °2θ positions 5.0±0.2, 5.1±0.2 and/or 11.8±0.2.
 12. The mixedsolvate according to claim 11, having an XRPD pattern, obtained withCuKα1-radiation, with peaks at °2θ positions 5.0±0.2, 5.1±0.2, 6.4±0.2,6.6±0.2, 9.5±0.2 and 11.8±0.2.
 13. The mixed solvate according to claim8, having an XRPD pattern, obtained with CuKα1-radiation, with peaks at°2θ positions 4.8±0.2, 5.1±0.2 and/or 11.6±0.2.
 14. The mixed solvateaccording to claim 13, having an XRPD pattern, obtained withCuKα1-radiation, with peaks at °2θ positions 4.8±0.2, 5.1±0.2, 6.2±0.2,6.67±0.2, 9.5±0.2, 11.6±0.2 and 20.3±0.
 15. The mixed solvate accordingto claim 8, having an XRPD pattern, obtained with CuKα1-radiation, withpeaks at °2θ positions 5.0±0.2, 6.2±0.2, 9.4±0.2 and/or 23.9±0.2. 16.The mixed solvate according to claim 15, having an XRPD pattern,obtained with CuKα1-radiation, with peaks at °2θ positions 5.0±0.2,6.2±0.2, 9.4±0.2 and 23.9±0.2 and one or more of the characteristicpeaks: 11.5±0.2, 19.5±0.2 and 20.2±0.2.
 17. A process for thepreparation of crystal modification 1 of odevixibat, comprisingisolating crystal modification 2 of odevixibat from a solution ofodevixibat in a solvent mixture comprising water and an organic solventselected from the group consisting of methanol, ethanol, 2-propanol,acetone, acetonitrile, 1,4-dioxane, DMF and DMSO.
 18. The processaccording to claim 17, wherein the crystal modification 2 of odevixibatis crystal modification 2A of odevixibat.
 19. The process according toclaim 17, wherein crystal modification 2A of odevixibat is obtained froma mixture of water and ethanol.
 20. The process according to claim 19,wherein the ethanol content in the solvent mixture is about 55 to about75% (v/v).